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Kinetic Characterization of Catalysis by Oxalate Decarboxylase Using Membrane Inlet Mass Spectrometry

Permanent Link: http://ufdc.ufl.edu/UFE0043598/00001

Material Information

Title: Kinetic Characterization of Catalysis by Oxalate Decarboxylase Using Membrane Inlet Mass Spectrometry
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Moral, Mario Edgar G
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: mims -- oxalate -- oxdc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate Decarboxylase (OxDC) from Bacillus subtilis is a manganese (II)-containing enzyme which catalyzes the breakdown of monoprotonated oxalate to carbon dioxide and formate. A hallmark of the reaction is its reported dependence on oxygen, which is not consumed in the process. Nothing is known about how Mn(II) catalyzes the reaction, or how the oxygen cofactor even participates in catalysis. However, efforts on further studying the enzyme have been severely limited by the indirect and discontinuous nature of assays established to measure enzymatic activity. My work reports on the application of Membrane Inlet Mass Spectrometry (MIMS) on catalysis by OxDC to develop a quick, direct and continuous assay through the measurement of product CO2. This real-time alternative has shown to deliver comparable measurements in OxDC kinetics, and additional ways to investigate catalysis which were difficult to achieve in previously established methods. In my work, MIMS was also employed to assess the product inhibition of OxDC catalysis and determine the reversible inhibition of a structural analog of O2 - nitric oxide (NO). Formate was weakly inhibitory (Ki > 200 mM) while high levels of carbon dioxide had no effect on catalysis. Inhibition by NO was found to be uncompetitive with a micromolar Ki value comparable to the reported binding constant of O2 on this enzyme. Continuous wave Electron Paramagnetic Resonance (cw-EPR) measurements of the enzyme in the presence of NO showed no binding interaction between NO and catalytic manganese of OxDC. This may either suggest a binding interaction of NO with the other Mn(II) center, or an undiscovered binding pocket for NO (or O2) elsewhere on the enzyme. A systematic study on the catalytic effect of small anions has shown nitrite, azide, thiocyanate, and bicarbonate to be inhibitory. Inhibition constants for these anions were in the low millimolar range, with interestingly different modes of inhibition on the enzyme. Nitrite was uncompetitive; azide and thiocyanate were noncompetitive, and bicarbonate was competitive. These results show the complexity of OxDC catalysis and areas for further study in elucidating the catalytic mechanism of the enzyme. Other aspects of OxDC explored by this new assay are also described.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mario Edgar G Moral.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Richards, Nigel G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043598:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043598/00001

Material Information

Title: Kinetic Characterization of Catalysis by Oxalate Decarboxylase Using Membrane Inlet Mass Spectrometry
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Moral, Mario Edgar G
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: mims -- oxalate -- oxdc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Oxalate Decarboxylase (OxDC) from Bacillus subtilis is a manganese (II)-containing enzyme which catalyzes the breakdown of monoprotonated oxalate to carbon dioxide and formate. A hallmark of the reaction is its reported dependence on oxygen, which is not consumed in the process. Nothing is known about how Mn(II) catalyzes the reaction, or how the oxygen cofactor even participates in catalysis. However, efforts on further studying the enzyme have been severely limited by the indirect and discontinuous nature of assays established to measure enzymatic activity. My work reports on the application of Membrane Inlet Mass Spectrometry (MIMS) on catalysis by OxDC to develop a quick, direct and continuous assay through the measurement of product CO2. This real-time alternative has shown to deliver comparable measurements in OxDC kinetics, and additional ways to investigate catalysis which were difficult to achieve in previously established methods. In my work, MIMS was also employed to assess the product inhibition of OxDC catalysis and determine the reversible inhibition of a structural analog of O2 - nitric oxide (NO). Formate was weakly inhibitory (Ki > 200 mM) while high levels of carbon dioxide had no effect on catalysis. Inhibition by NO was found to be uncompetitive with a micromolar Ki value comparable to the reported binding constant of O2 on this enzyme. Continuous wave Electron Paramagnetic Resonance (cw-EPR) measurements of the enzyme in the presence of NO showed no binding interaction between NO and catalytic manganese of OxDC. This may either suggest a binding interaction of NO with the other Mn(II) center, or an undiscovered binding pocket for NO (or O2) elsewhere on the enzyme. A systematic study on the catalytic effect of small anions has shown nitrite, azide, thiocyanate, and bicarbonate to be inhibitory. Inhibition constants for these anions were in the low millimolar range, with interestingly different modes of inhibition on the enzyme. Nitrite was uncompetitive; azide and thiocyanate were noncompetitive, and bicarbonate was competitive. These results show the complexity of OxDC catalysis and areas for further study in elucidating the catalytic mechanism of the enzyme. Other aspects of OxDC explored by this new assay are also described.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mario Edgar G Moral.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Richards, Nigel G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043598:00001


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1 KINETIC CHARACTERIZATION OF CATALYSIS BY OXAL ATE DECARBOXYLASE USING MEMBRANE INLET MASS SPECTROMETRY By MARIO EDGAR GABUA MORAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Mario Edgar Gabua Moral

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3 To my parents my sister, and my late grandparents

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4 ACKNOWLEDGMENTS Th is project was supported by grants from th e N ational Institutes of Health (NIH DK061666 & GM25154 ) and the National Science Foundation ( CHE 080972 5). I would like to thank my committee members : Dr. David Silverman for the use of the modified Membrane Inlet Mass Spectrometer in his lab; Dr. Alex An gerhofer for the continued support on the EPR side of the project ; Dr Nicole Horenstein ; and Dr. Gail Fanucci for all the insights and useful discussions on enzyme kinetics and physical chemistry; and most especially my research adviser Dr. Nigel G. J. Richards, for providing me a part in the oxalate decarboxylase project and in his coll aborations which got us to where we are now. I owe a debt of gratitude to Dr. Chin g kuang Tu for the fruitful collaboration behind our MIMS experiments and for inspiring my creativity in research throu gh his innovativeness and insight on every scientific problem we had to process. I would like to acknowledge Dr. Stephan Bornemann and his research group at the Joh n Innes Institute in Norwich, U.K. for generously providing t he plasmid containing the gene for the C terminal polyhistidine tagged ( His6 tagged ) OxDC from Bacillus subtilis I am deeply gr ateful for my former research colleagues: Dr. Patricia Moussatche, Dr. Ellen Moomaw and Erin Holmes for the invaluable skills I learned in the beginning of my research. I thank Dr. Witcha Imaram for his assistance with the EPR experiments and Ms. Mithila Shukla for the characteriz ation and generous provision of the tyrosine OxDC mutant Y200F I especially would like to acknowledg e the generosity of Dr. Giovanni Gadda and Dr. Kevin Francis of Georgia State University for the use of their Hansatech oxygen electrode in the early stages of this project

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5 I thank Dr. Cris Dancel for recruiting me to the graduate chemistry program of UF Dr s. Dodge and Jhoana Baluya, Lilibeth Salvador, Carmello Callueng, Benjamin Raterman, Dr. Mandy Blackburn Dr. Modesto Chua, Dr. Flerida Carino, and Dr. Florecita de Guzman for all the encouragement and invaluable support throughout my graduate school. F inally, I thank my sister Diane, my pa rents and late grandparents for instilling in me the value of hard work, perseverance, and faith.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 16 Bacillus subtilis Oxalate Decarboxylase ( Bs OxDC) Structure and Reaction ........... 16 Decarboxylase Activity Assays ................................ ................................ ............... 22 Formate Dehydrogenase (FDH) Coupled Assay ................................ .............. 22 Fourier Transform Infrared (FTIR) Spectrophotometric Assay ......................... 23 Limitations of Current Decarboxylase Activity Assays ................................ ...... 24 Membrane Inlet Mass Spectrometry (MIMS) ................................ .......................... 25 Research Objectives ................................ ................................ ............................... 26 2 CHARACTERIZATION OF OXDC BY MEMBRANE INLET MASS SPECTRO METRY ................................ ................................ ................................ .. 27 Introduction ................................ ................................ ................................ ............. 27 Reaction Vessel ................................ ................................ ............................... 27 Membrane Inlet Probe and (Electron Impact) Ionization ................................ .. 28 Mass Analyzer ................................ ................................ ................................ .. 29 Results and Discussion ................................ ................................ ........................... 31 Real time Monitoring of catalysis ................................ ................................ ...... 31 Reproducibility ................................ ................................ ........................... 35 Response time ................................ ................................ ........................... 36 Buffer conditions: Air equilibrated vs. O 2 depleted (He bubbled) ............... 36 Background CO 2 and Isotope labeled substrate ( 13 C 2 oxalate) ................. 38 o Phenylenediamine (o PDA) ................................ ................................ .... 39 Calibration ................................ ................................ ................................ .. 39 Data Analysis: Michaelis Menten Kinetics ................................ ........................ 42 Steady state ................................ ................................ ............................... 42 Initial velocities ................................ ................................ ........................... 43 Sources of error ................................ ................................ ......................... 44 Kinetic constants (MIMS vs FDH) ................................ .............................. 45 Experimental Section ................................ ................................ .............................. 48 Materials ................................ ................................ ................................ ........... 48

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7 His 6 tagged Oxalate Decarboxylase Expression and Purification .................... 49 Formate Dehydrogenase (FDH) Coupled Activity Assay ................................ 50 Membrane Inlet Mass Spectrometry (MIMS) ................................ .................... 50 Reaction Mixtures ................................ ................................ ............................. 51 3 NITRIC OXIDE INH IBITION OF CATALYSIS BY OXDC ................................ ........ 53 Introduction ................................ ................................ ................................ ............. 53 Nitric Oxide as Dioxygen Mimic ................................ ................................ ........ 53 Nitric Oxide from NONOates ................................ ................................ ............ 53 Data Analysis: Inhibition ................................ ................................ ................... 55 Results and Discussion ................................ ................................ ........................... 61 Reversible Inhibition of NO ................................ ................................ ............... 61 OxDC inhibition by DEA NONOate ................................ ............................ 65 Dependence of OxDC inhib ition on NONOate concentration ..................... 66 Mode of Inhibition of NO ................................ ................................ ............ 66 Inhibition of NO on catalysis by site specific mutants of OxDC .................. 68 Constant Wave Electron Paramagnetic Resonance (CW EPR) Experiments .. 72 Experimental Section ................................ ................................ .............................. 74 Materials ................................ ................................ ................................ ........... 74 His6 tagged Oxalate Decarboxylase Expression and Purification .................... 74 Untagged Recombinant Wild type and C383S OxDC Mutant .......................... 75 Membrane Inlet Mass Spectrometry (MIMS) ................................ .................... 75 Reaction Mixtures ................................ ................................ ............................. 75 Continuous Wave EPR Measurements ................................ ............................ 76 4 SMALL ANION INHIBITION OF CATALYSIS BY OXDC ................................ ........ 79 Introdu ction ................................ ................................ ................................ ............. 79 Results and Discussion ................................ ................................ ........................... 79 Catalytic Products ................................ ................................ ............................ 80 Anionic Buffer Effects ................................ ................................ ....................... 82 Nitrate and Nitrite ................................ ................................ ............................. 83 Bicarbonate ................................ ................................ ................................ ...... 84 Azide a nd Thiocyanate ................................ ................................ ..................... 86 Experimental Section ................................ ................................ .............................. 89 His6 tagged Oxalate Decarboxylase Expression and Purification .................... 89 Membrane Inlet Mass Spectrometry (MIMS) ................................ .................... 89 Reaction Mixtures ................................ ................................ ............................. 89 Anion Inhibition ................................ ................................ ................................ 89 Initial Rates ................................ ................................ ................................ ....... 90 Mode of Inhibition and Statistical Estimation of Parameters ............................. 90 5 CONCLUSIONS AND FUTURE WORK ................................ ................................ 91 MIMS and OxDC Catalysis ................................ ................................ ..................... 91 Insights on Future Work ................................ ................................ .......................... 92

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8 Michaelis Menten Complex of OxDC ................................ ................................ 92 Oxygen binding site and Oxygen Dependence ................................ ................ 93 MIMS and Site specific Mutants ................................ ................................ ....... 94 Effect of Periodate and biSulfite anions ................................ ............................ 95 APPENDIX A EXPRESSION AND PURIFICATION OF BACILLUS SUBTILI S OXDC ................. 96 B OXDC SEQUENCES AND ALIGNMENTS ................................ ............................. 99 LIST OF REFERENCES ................................ ................................ ............................. 102 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 108

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9 LIST OF TABLES Table page 2 1 Steady state constants for decarboxylation of oxalate catalyzed by polyhistidine tagged OxDC measured by MIMS and by for mate dehydrgenase coupled assay ................................ ................................ ............. 47 3 1 Table of comparison between graphical features of Lineweaver Burke and Hanes plots. ................................ ................................ ................................ ........ 61 3 2 Modes of Inhibition represented by L ineweaver Burke and Hanes Plots ............ 61

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10 LIST OF FIGURES Figure page 1 1 Reactions cataly zed by the three class es of oxalate degrading enzymes ......... 16 1 2 Ribbon structure of germin oxalate oxidase from Hordeum vulgare ................. 17 1 3 Ribbon structure of B. subtilis oxalate decarboxylase hexa mer, trimer, and monomeric unit ................................ ................................ ................................ .. 18 1 4 Partial seque nce alignment of OxDC with OxOx showing the conserved active site lid glut amate in OxDC which is not present in OxOx. ........................ 18 1 5 Mn(II) centers of the B subtilis OxDC monomer and the conserved set of one glutamate and three histidine ligand binding residues.. ................................ ...... 19 1 6 Proposed mechanism of the B. subtilis OxDC catal yzed decarboxylation of oxalate ................................ ................................ ................................ ............... 20 1 7 Coupling reaction of the FDH Assay for oxal ate decarboxylases. ...................... 23 2 1 The membrane inlet inserted in an air tight cell for mass spectrometric measurements. ................................ ................................ ................................ ... 27 2 2 Picture of the modified MIMS instrument and b lock diagram illustrating the main components of the system. ................................ ................................ ........ 28 2 3 Cartoon showing how the time dependent growth in peak height of a selected signal is r eillustrated as a progress curve on the monitor of the MIMS instrument. ................................ ................................ ............................... 30 2 4 Experimental scheme for air equilibrated catalytic experiments.. ....................... 32 2 5 Ion currents (arbitrary scale) in the production of carbo n dioxide from 12 C and 13 C 2 labeled oxalate catalyzed by OxDC in air equilibrated buffer.. .................... 32 2 6 Ion current in the production of 13 CO 2 from 13 C 2 oxalate catalyzed by OxDC. .... 35 2 7 Superimposed ion currents (arbitrary scale) of three separate experiments involving the production of 13 CO 2 from 13 C 2 oxala te catalyzed by OxDC.. .......... 36 2 8 Experimental scheme for oxygen depleted catalytic experiments ..................... 37 2 9 Ion currents in the produ cti on of carbon dioxide from 12 C and 13 C 2 labeled oxalate potassium oxalate catalyzed by OxDC in deoxygenated buffer. ............ 37 2 10 Overlay of m/z 44 ion currents illustrating background CO 2 detected f rom the air equilibrated OxDC enzyme aliquot in the absence of substrate ................... 39

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11 2 11 Ion current (arbitrary scale) at m/z 44 using the membrane inlet mass spectrometer observed using solutions of known CO 2 content.. ......................... 40 2 12 Ion current (arbitrary scale) at m/z 32 using the membrane inlet mass spectrometer observed using solutions of calculated O 2 content ...................... 41 2 13 Example of deriving initial rates from a progress curve of CO 2 formation ........... 44 2 14 Accumulation of 13 CO 2 in solution result ing from the catalysis by Ox DC. ........... 45 2 15 Initial rates of the appearance of 13 CO 2 in solutions resulting from catalysis of the decarboxylation of oxalate by OxDC. ................................ ........................... 46 2 16 Initial rates of catalysis by polyhistidine tagged B. subtilis oxalate decarboxylase determined by the formate dehydrogenase coupled endpoint assay ................................ ................................ ................................ .................. 48 3 1 Structures of MAHMA / D EA NONOates at pH 8.5, and their respective reaction byproducts at pH 7 ................................ ................................ .............. 54 3 2 Modified experimental scheme for testing the effects of NO on catalysis by OxDC. ................................ ................................ ................................ ................. 55 3 3 Fundamental enzymatic reaction model. ................................ ............................ 57 3 4 Enzymatic scheme for competitive inhibition. ................................ ..................... 57 3 5 Enzymatic scheme for uncompetitive inhibition. ................................ ................. 58 3 6 Enzymatic scheme for noncompetitive/mixed inhibition ................................ ...... 59 3 7 Effec t of NO on catalysis by C terminally His 6 tagged OxDC. ............................ 62 3 8 Effect of N 2 on catalytic inhibition by NO ................................ ........................... 63 3 9 MIMS experiment showing the effect of adding MAHMA NONOate to OxDC during catalytic turnover. ................................ ................................ .................... 64 3 10 MIMS experiments showing that OxDC inhibition is not dependent on the source of NO ................................ ................................ ................................ ..... 65 3 11 Dependence of OxDC inhibition on initi al MAHMA NONOate concentration. ..... 66 3 12 Mode of Inhibition of NO. ................................ ................................ .................... 67 3 13 Effect of NO on catalysis by untagged C383S OxDC mutant ............................ 68 3 14 Effect of NO on catalysis by Y200F OxDC mutant. ................................ ............ 69

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12 3 15 Effect of hemoglobin on catalytic inhibition by NO. ................................ ............. 70 3 16 Effect of hemoglobin on catalytic inhibition by NO after bubbling with N 2 .......... 71 3 17 Overlaid CW EPR spectra of the Mn (II) centers in OxDC in the presence and absence of NO released from MAHMA NONOate. ................................ ...... 72 4 1 Inhibition by azide ions of catalysis by OxDC. ................................ .................... 80 4 2 Effects of dissolved halides on catalysis by OxDC ................................ ............ 81 4 3 Inhibition by nitrite ion of catalysis b y OxDC. ................................ ...................... 83 4 4 Inhibition by bicarbonate of catalysis by OxDC. ................................ .................. 85 4 5 Mode of inhibitio n by azide of catalysis by OxDC ................................ ............... 87 A 1 12% SDS PAGE gel of fractions from the expression and purification of C terminally His 6 tagged wild type Bs OxDC. ................................ .......................... 97 A 2 12% SDS PA GE gel of fractions from the expression and purification of untagged wild type Bs OxDC. ................................ ................................ .............. 97 A 3 12% SDS PAGE gel of fractions from the expression and purification of untagged C383S Bs OxDC mutant ................................ ................................ ..... 98 B 1 Amino acid sequence alignment of several bacterial and fungal OxDC using ClustalW. ................................ ................................ ................................ ............ 99 B 2 Amino acid sequence alignment of bac terial and fungal OxDC with the bicupin ox alate oxidase (OxOx) from yeast ................................ ...................... 100 B 3 Amino acid and yvrk gene sequences of oxalate decarboxylase from B. subtilis. ................................ ................................ ................................ ............ 101

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13 LIST OF ABBREVIATION S AC a lternating current DC d irect current DEA NONOate diethylammonium (Z) (N,N diethyl amino)diazen 1 ium 1,2 diolate FDH formate dehydrogenase HCO 3 bicarbonate anion His6 tagged protein bearing a polyhistidine affinity t ag comprised of six histidine residues MAHMA NONOate (Z) 1 (N methyl N [6 (N methylammoniohexyl)amino]diazen 1 ium 1,2 diolate MIMS membrane inlet mass spectrometry MnSOD manganese superoxide dismutase N 3 azide anion NAD+ nicotinamide adenine dinucleotide Ni NTA nickel nitrilotriacetic acid NO nitric oxide NO 2 nitrite anion NO 3 nitrate anion O PDA ortho phenylenediamine OxDC oxalate decarboxylase SCN thiocyanate anion SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

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14 Abstract of Disse rtation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy KINETIC CHARACTERIZATION OF CATALYSIS BY OXALATE DECARBOXYLASE USING MEMBRANE INLET MA SS SPECTROMETRY By Mario Edgar Gabua Moral December 2011 Chair: N igel G. J. Richards Major: Chemistry Oxalate Decarboxylase (OxDC) from Bacillus subtilis is a manganese (II) containing enzyme which catalyzes the breakdown of monoprotonated oxalate to ca rbon dioxide and formate. A hallmark of the reaction is its reported dependence on oxygen, which is not consumed in the process. Nothing is known about how Mn(II) catalyzes the reaction, or how the oxygen cofactor even participates in catalysis. However, e fforts on further studying the enzyme have been severely limited by the indirect and discontinuous nature of assays established to measure enzymatic activity. My work reports on the application of Membrane Inlet Mass Spectrometry (MIMS) on catalysis by OxD C to develop a quick, direct and continuous assay through the measurement of product CO 2 This real time alternative has shown to deliver comparable measurements in OxDC kinetics, and additional ways to investigate catalysis which were difficult to achieve in previously established methods. In my work, MIMS was also employed to assess the product inhibition of OxDC catalysis and determine the reversible inhibition of a structural analog of O 2 nitric oxide (NO). Formate was weakly inhibitory (K i > 200 mM) while high levels of carbon dioxide had no effect on catalysis. Inhibition by NO was found to be uncompetitive with

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15 a micromolar K i value comparable to the reported bin ding constant of O 2 on this enzyme. Continuous wave Electron Paramagnetic Resonance (cw EPR) measurements of the enzyme in the presence of NO showed no binding interaction between NO and catalytic manganese of OxDC. This may either suggest a binding interaction of NO with the other Mn(II) center, or an undiscovered binding pocket for NO (or O 2 ) elsewhere on the enzyme. A systematic study on the catalytic effect of small anions has shown nitrite, azide, thiocyanate, and bicarbonate to be inhibitory. Inhibition constants for these anions were in the low millimolar range, with interestingly diff erent modes of inhibition on the enzyme. Nitrite was uncompetitive; azide and thiocyanate were noncompetitive, and bicarbonate was competitive These results show the complexity of OxDC catalysis and areas for further study in elucidating the catalytic mec hanism of the enzyme. Other aspects of OxDC explored by this new assay are also described.

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16 CHAPTER 1 BACKGROUND Bacillus subtilis Oxalate Decarboxylase ( Bs OxDC) Structure and Reaction Oxalate decarboxylases are one of three classes of oxalate degrading e nzymes which are found in fungi and bacteria. The other two classes are either found mostly in plants (oxalate oxidases), or bacteria (Oxalyl CoA decarboxylases). (A) (B) (C) Figure 1 1. Reactions catalyzed by the three classes of oxalate degrading enzymes. Oxalate degraded by (A) oxalate decarboxylases are found mostly in fungi and bacteria, (B) oxalate oxidases are found mostly in plants, while (C) Ox alyl CoA decarboxylase are found in bacteria. Oxalate decarboxylase s catalyze the breakdown of monoprotonated oxalate to formate and carbon dioxide Figure 1 1A ( 1 5 ) C atalysis is believed to be aerobic, though the required oxygen is not consumed in the reaction ( 1 4 ) This non oxidative reaction feature of oxalate decarboxylase contrasts from that of a closely related class of enzyme oxalate oxidase, which shares the same o xalate substrate and requirement for dioxygen for catalysis, but in con trast, oxidatively breaks down monoprotonated oxalate into an additional molecule of carbon dioxide and hydrogen peroxide (Figure 1 1B) ( 5, 6 ) T he striking similarity between the structures of oxalate decarboxylase and

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17 oxidase ( Figure s 1 2 and 1 3 ) has strongly suggested an evolutionary relationship between the two enzymes. This idea was supported by the fact that the oxalate decarboxylase from so il bacterium B. subtilis was observed to have a small (0.2 0.4%) inherent oxidase activity amidst its more predominant decarboxylase activity ( 7 ) (A) (B) Figure 1 2. Ribbon structure of germin oxalate oxidase from Hordeum vulgare (8) Colored spheres are respective Mn(II) atoms in the (A) homohexamer comprised of a trimer of dimeric units. (B) monomeric unit is a single domain defined by a barrel fold containing a Mn(II) atom. PDB: 1FI2. Structures were generated using P yMol v0.99 DeLano Scientific (San Francisco, CA). B. subtilis oxalate decarboxylase crystallizes as a hexamer composed of a dimer of trimeric units ( Figure 1 3 a b ). Each monomeric unit is characterized to have two Mn(II) atoms, each held in place by coord ination with the sidechains of a conserved set of one glutamate and three histidine residues ( 5, 9, 10 ) Each of these manganese barrel motif, referred to as a cupin fold ( 5, 11 13 ) giving rise to an N and C terminal manganese site in every bicupin monomer. Various work in literature involving site directed mutagenesis and electron paramagnetic resonance (EPR) have suggested the N terminal Mn(II) s ite to be the solvent accessible and main catalytic site in the enzyme ( 14 18 )

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18 (A ) (B ) (C ) Figure 1 3 Ribbon structure of B subtilis o xalate d ecarboxylase hexamer, trimer, and monomeric unit. (A) Side vie w of the BsOxDC hexamer revealing a dimer of trimeric units (B) stacked in a back to back fashion. (C) Monomeric unit comprised of a symmetric set of two barrel folds, which define two domains within each holds a Mn(II) atom. PDB: IJ58 Structures were g enerated using PyMol v0.99 DeLano Scientific (San Francisco, CA). The structural similarities shared between the active sites of oxalate decarboxylases and its close relative oxalate oxidases, have not only implied the evolutionary relationship between t hem ( 11, 19, 20 ) ; but also facilitated the discovery of a five residue N in B. subtilis oxalate decarboxylases ( 14 ) On as found a vital protonating glutama te residue Glu162 (Figure 1 4) which did not exist in the structurally similar oxalate oxidase ( 12, 14, 21 ) B. subtilis OxDC 160 F S E NST F B. amyloliquofasciens OxD C 166 F S E NST F A. niger OxDC 228 F S E EST F C. subvermispora OxOx 240 FDASNQF H. vulgare OxOx 54 EAGDDF Figure 1 4 Partial sequence alignment of bacterial and fungal OxDC with yeast and plant OxOxshowing the cons erved glutamate (blue) on a presumed lid sequence (red) in OxDC ( 14 ) which is absent in OxOx.

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19 Mo dels on the m bility of this five conformational cha nges in this loop, which were believed to regulate solvent access to the active site, and the proper positioning of the conserved glutamate lid residue for its role in catalysis ( 10 ) (Figure 1 5 ) Literature suggest s that catalysis in oxalate decarboxylase takes place when this N suggesting that substrate access to the active site and product release occur only in the ( 9, 10, 14, 15 ) Figure 1 5 Mn(II) centers of the B subtilis OxDC monomer and the conserved set of one glutamate and three histidine ligand binding residues Research was originally published in ( 10 ) A merican Society for Biochemistry and Molecular Biology ( Top l eft) N terminal showing the octahedral coordinated Mn(II), catalytic Glu 162 and what was interpreted to be formate from the electron density uniquely observe d in this Mn(II) center. Independent red spheres are water molecules. ( Top r ight) C terminal Mn (II) with a symmetric set of residues around the similarly coordinated metal. PDB: IL3J. (Bottom left) N conformation, where G lu 162 is now in closer proximity to serve as a proton donor/acceptor to presumed oxalate bound to Mn(II) at one of the two available slots occupied in the model by water molecules (red spheres)

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20 The mechanism of catalysis in this enzyme is not yet fully e lucidated, but density functional theory (DFT) calculations and experiments employing kinetic isotope effects have led to the currently proposed mechanism of the catalyzed decarboxylation, which suggests a proton coupled electron transfer (PCET) ( 5, 22, 23 ) In this mechanism, the dioxygen cofactor binds to manganese, which becomes the electron transfer hub between dioxygen and the bound substrate. The Mn bound monoprotonated oxalate substrate is polarized by the int eraction of one of its C=O bonds with a conserved arginine ( 21 ) basic residue glutamate 162, which results in the heterolytic C C cleavage ( 5, 18, 23 ) and departure of CO 2 This cleavage results in a Mn bound formate radical. This bound formate radical intermediate is believed to be reprotonated by the same glutamate 162 residue ( 5, 21, 23 ) presumably i Glu 162 is positioned closest to the hypothesized radical intermediate in the interior of the manganese active site ( 10, 15 ) Figure 1 6 Proposed mechanis m of the B subtilis OxDC catalyzed decarboxylation of oxalate ( 23 )

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21 This hypothesis on glutamate 162 is consistent with site directed mutagenesis work done on this residue and on other lid residues, where the replacement of Glu 162 abolished decarboxylase activity in the enzyme. Furthermore, three additional amino acid substitutions n the chemistry of the enzyme towards promoting oxidase activity. This remarkable result supplemented the proposed catalytic mechanism in the enzyme, implicating Glu 162 to be the vital residue controlling the fate of the radical intermediate ( 24 ) in oxalate decarboxylase (Figure 1 6 ) towards being protonated and released in the form of formate, rather than oxidized into hydrogen peroxide and CO 2 (oxidase path) ( 14, 21, 23 ) T he fact that four additional am ino acid substitutions on the lid other than Glu 162 were required to promote the specificity switch from decarboxylase to oxidase further indicates that overall protein environment in the active site also has a vital role in influencing the chemistry of a n ( 14 ) Other site directed mutagenesis work ha d established the dependence of decarboxylase activity on the amount of manga nese incorporated in the enzyme. These studies not only supported earlier claims on the requirement of manganese in expressing th e enzyme but also suggest ed the possibility of the C terminal manganese site contributing to the catalytic properties of the assumed main active site in the N terminal domain ( 18 ) Despite considerable progress in literature, other structural features of the enzyme such as those responsible for accommodating the required dioxygen in the c atalytic reaction remain unknown Co ntinued efforts in studying this enzyme are largely guided by a close monitoring of enzymatic activity, which is often correlated with

PAGE 22

22 c ontrolled modifications on site specific enzyme residues, or on catalytic reaction conditions. D ecarboxylase Activity Assays For mate Dehydrogenase (FDH) Coupled Assay De c a rboxylase activity has most often been measured by a traditional endpoint assay, which involv es two main reactions : ( I ) the formate generating OxDC catalytic reaction (F igure 1 1A ) and ( II ) a coupling reaction wh ich degrades formate with the concomitant reduction of nicotinamide adenosine din ucleotide (NAD+) to NADH (F igure 1 7 ) ( 21, 25, 26 ) The first reaction is usually conducted in a semi micro vessel (e.g. eppendorf tub e) containing enzyme stabilizing components which are buffered with sodium acetate at catalytic optimum pH of 4.2 and 25 o C. Cataly sis is initiated by the addition of enzyme or substrate and then quenched after a predetermined amount of time by the addition of at least 100 mM sodium hydroxide base. Addition of the strong base raises the solution pH resulting in the complete deprotonation of unreacted oxalate substrate in solution. Alth ough unprotonated oxalate is no longer viable for further catalysis, the OxDC enzyme in solution remains active at this elevated pH. Amount of catalytic formate formed in the abovementioned reaction is quantified in a separate reaction where an aliquot of the earlier quenched reaction mixture is incubated overnight i n a solution of nicotinamide adenosine dinucleotide (NAD+) and formate dehydrogenase, buffere d at pH 7.6 with potassium phosphate. The quantity of reduced NAD+ (NADH) from the breakdown of formate is measured spectrophotometrically at 340 nM ( 21, 25, 26 ) and calibrated against absorbances of similarly treated solutions containing known amounts of pre formed formate.

PAGE 23

23 Figure 1 7 Coupling reaction of the FDH Assay for oxalate decarbox ylases. Formate is converted to carbon dioxide with a concomitant reducti on of adenosine nicotinamide di nucleotide to a form in solution which is detected by its measured absorbance at 340 nm. Fourier Transform Infrared (FTIR) Spectrophotometric Assay T he first real time monitoring of decarboxylase activity reported involved the use of fourier transform infrared spectrophotometry ( 27 ) This method took advantage of the infrared absorption of substrate and products, which were admittedly rare properties had the ability to simultaneously monitor the consumption of oxalate and the formation of carbon dioxide, carbonate, and formate The method is based on time dependent changes in a bsorbance s a ssociated with the C O bond vibrations of substrate and product throughout a single catalytic reaction. These different C O bond bend/stretches absorb at distinct regions on the 1000 4000 cm 1 scanning range, which is sampled in 25 millisecond intervals ov er a period of 5 minutes. Ability of this method to monitor both oxalate and product formate has e nabled this approach to also monitor oxidase activity in OxDC and mutant enzyme constructs. Since decarboxylase activity is indicated by the loss of oxa late ( 1308 cm 1 ) concomitant to the emergence of formate (1385 and 1352 cm 1 ) any loss of oxalate which is not accompanied by the emergence of peaks associated with formate product would indicate the alternative route in catalysis most c oherent with oxidase act ivity. The method has also been employed in investigating solvent interactions with catalysis and

PAGE 24

24 the fate of substrate atoms by the use of isotope labeled reagents These were accomplished by monitoring expected shifts in FTIR absorbances associated with modified bond stretches from heavier isotopes incorporated in catalytic products. Limitations of Current Decarboxylase Activity Assays Under optimum conditions, specific activity and kinetic measurements on wild type OxDC may conveniently be accomplished u sing the FDH coupled endpoint assay. The same can be said for type behavior This is because t imecourses (0.5 1 minute) for the first catalytic reaction have previously been optimized to reliab ly sample the initial rate s of product formation prior to the FDH coupled reaction step when these are quantified The inability to acquire catalytic progress curves from a single experiment presents a problem especially when reaction conditions change the timeframe whe n initial r ates of the enzyme ( or mutant ) are best measured. Separate timecourse experiments may be designed to survey the best timeframe to run the f irst catalytic reaction, but this preliminary step entails a long turnaround time and numerous sample preparations. F urthermore, any experimental conditions which affect the FDH coupling reaction instantly render s this assay useless in studying OxDC. Real time monitoring of catalysis by FTIR spectrophotometry not only provide s the first cont inuous assay for OxDC, but als o the versatility of monitoring decarboxylase and oxidase activity However, d ata processing involves the deconvolution of accumulated spectra which can both be tedious and time consuming. Ability to do experiments involving isotope labeled compounds is a great advantage in this method. However, shifts in absorbances may not always be simple to track down, especially for reaction

PAGE 25

25 conditions involving substances which may also have absorbances in the same region of detection. In both assays detection limits are an issue. Re ported UV absorbances for example, are only re liable when they are at a magnitude within the linear region of detection This often translates to the need for experimental conditions where considerable amounts of product have to be generated. These conditions are difficult to attain when working with weakly active mutant enzyme constructs or when studying inhibitors. Membrane Inlet Mass Spectrometry (MIMS) Biochemical applications of mass spectrometry have been emerging in fi elds of proteomics, metabolomics, and medical research. Interfaced a nalytical devices such as high performance liquid chromatograph s (HPLC) as inlet s to a mass spectrometer ( 28 ) have provide d the ability to isolate and quantify component s of mixtures w hile subsequently identif ying them through precis e mass measurements ( 29, 30 ) A much simpler inlet utilizing a semipermeable membrane to select species from a solution or suspension to enter a mass spectrometer was first demonstrated by Hoch and Kok ( 31 ) The usefulness of the method in measuring gases in physiological studies of algae and plants had set the precedent for diversified applications of membrane inlets in many st udies employing mass spectrometry Biological applications of m embrane i nlet m ass s pectrometry (MIMS) is well described in the measurement of CO 2 in physiological samples ( 32, 33 ) volatile organic compounds ( 34 ) and in understanding enzymatic mechanism s ( 35 ) Membrane inlets in these studies were made of materials which were permeable to non polar and low molecular weight molecules Because these membrane inlets can be immersed in a solution for direct measurement of

PAGE 26

26 dissolved gases in solution, they are feasible for use in gas evolving enzym atic reaction mixtures. Thou gh MIMS has been employed in the study of certain enzymes, it has not been applied to the study of production of CO 2 in catalysis by the decarboxylases Research Objectives M easurement of catalytic activity is vital to the study of any enzyme Current assays for oxalate decarboxylase activity may provide dependable routine kinetic measurements for protein preparati ons, but they lack the versatility to further experiments in studying OxDC. A quick, yet sensitive continuous assay w hich can support a broader range of reaction conditions may provide more information to validate long standing hypotheses on the OxD C enzyme, its mechanism, and its potential applications The main objectives of the presented research were: 1) t o develop a n alternatively quick, direct, and continuous assay employing MIMS on catalysis by OxDC ; 2) to utilize MIMS to fu rther investigate the OxDC catalytic reaction ; and, 3) t o survey and characterize inhibitors of OxDC by MIMS to gather more information for fut ure experiments exploring medical applications of the enzyme and its catalytic forms i n its reaction mechanism

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27 CHAPTER 2 CHARACTERIZATION OF OXDC BY MEMBRANE INL ET MASS SPECTROMETRY Introduction MIMS uses diffusion across a membrane as an inlet to a mass spectrometer (Fig ure 2 1) Because the method is based on the permeability of the membrane inlet to dissolved gases while being mostly impermeable to water and charged solutes, catalytic generation of carbon dioxide in solution by OxDC can provide a good basis for measuring decarboxylase activity through MIMS. Figure 2 1. T he membrane inlet inserted in an air tight cell for mass spectrometric measurements: 1) tubing leads to mass spectrometer; 2) sample introduction port with septum; 3) threaded glass port for connecting to vacuum or for introduction of inert gas; 4) vacuum needle valve stopcock; 5) wire helix for support of the Silastic tubing; 6) Silastic tubing of length 1 cm (1.5 mm i.d. and 2.0 mm o.d.); 7) glass bead to seal the Silastic tubing; 8) glass optical cuvette; 9) magnetic stirring bar ( 35 ) Reprinted from ( 36 ) with permission. Reaction Vessel Reaction mixtures (2 mLs) are placed in a fabricated quartz cuvette which is jacketed in a circulating water bath, whose temperature may be regulated for desired constant temperature conditions. The vessel contains a magnetic stir ring bar which

PAGE 28

28 constantly stirs the reaction mixture on a supporti ng magnetic stirplate. The reaction chamber has a minimum of three gas tight inlets/caps: one for the membrane inlet probe (interfaced to the mass spec); one for the (air/helium) gas inlet and outlet for equilibrating reaction mixtures, and one for the add ition of sample or buffer components. Figure 2 2 Picture of the modified MIMS instrument (Bottom) Block diagram illustrating the main components and schematic of the system Membrane Inlet Probe and (Electron Impact) Ionization The reaction chamber is interfaced with the instrument through a membrane inlet probe immersed in the reaction solution. The tip of the immersed membrane inlet probe is equipped with a piece of inert silicone tubing (Dow Corning), which is permeable to small uncharged/hydroph obic species. The open end of the tube is plugged with a glass bead and the length of the tubing is supported by an inner coil, which serves as an inner

PAGE 29

29 scaffold preventing the flexible tubing from collapsing under partial vacuum on the tip of the probe. T he resulting cavity within the cylindrical tubing allows membrane selected species into the probe and into the ionization chamber of the instrument. The path between the membrane and the instrument is routed through a cold trap of dry ice and acetone. Thi s cold trap freezes out any water vapor in the lines, preventing water molecules from entering the mass spec instrument. Since detection of species are dependent on the successful flight of molecules through the instrument, rationalizing the need to maint ain the lines under a certain vacuum, unwanted water molecules in the instrument may interfere in the flight path of the species and thus adversely affect overall signal detection. Gaseous molecules passing through the inlet travel to the ionizer where a b eam of electrons collide with the influx of sample molecules. Electrons in the beam collide with each molecule. This process results in a population of positive ioni c species which proceed into the linear quadrupole, where they are differentiated by their relative masses. Mass Analyzer The mass analyzer in this instrument is a single quadrupole comprised of four rod electrodes in a square configuration ( 28 ) with a small central gap serving as the inner rod electrode has an a lternatin g polarity assignment resulting from a potential composed of a DC and AC component ( 28 ) Since the ionized specie would be att racted to a rod of opposite charge, it is conceivable that the ion will crash against one of the four rods it is attracted to. However, the polarity assignments of each rod periodically toggle at a certain

PAGE 30

30 frequency, allowing the successful passage of the ion through the central cavity of the travels through the mass analyzer is directly associated with its ionic mass, which is programmed Th us, the quadrupole varies the frequency at which it toggles the polarity assignments, resulting in a frequency sweep that continuously scans for ionic species from larger masses to lower masses throughout the experiment Ionic species which successfully tr avel through the quadrupole are detected upon their contact to a detector plate which results in an ion current that is recorded by the instrument. These mass differentiated populations of detected ions are recorded and traditionally reported as peaks on a com puter monitor. This instrument has an additional software which periodically samples a point from a defined vertical coordinate at each selected m/z peak and simultaneously plo ts these points on a time course. These time course plots result in progress curves (for each selected signal) describing the reaction profile occurring in the vessel throughout a catalytic experiment. Figure 2 3 illustrates the real time representation of a single m/z signal on a time course plot. A) B) Figure 2 3 Cartoon sh owing how the (A) time dependent growth in peak height of a selected signal is reillustrated as a (B) progress curve on the monitor of the MIMS instrument.

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31 In demonstrating the application of MIMS to kinetic studies of decarboxylases, decarboxylation of 12 C and 13 C 2 labeled oxalate was measured to yield 12 CO 2 13 CO 2 and formate catalyzed by OxDC from B. subtilis Dissolved gases were detected by immersing the membrane inlet into the air equilibrated reaction solution and monitoring different masses using t he ion current at peak heights. Reactions were carried out in an air tight vessel such as shown in Figure 2 1. The immersed membrane inlet which was permeable only to small uncharged species, was interfaced to a mass spectrometer through a cold trap of dr y ice and acetone. Gaseous molecules entering the probe were ionized in the mass spectrometer by electron impact (EI), and analyzed through a standard linear quadrupole (Figure 2 2) Detected ions were recorded by their mass to charge (m/z) ion currents, which n umerically correspond ed to their respective molecular weights. Time dependent changes in m/z peak heights we re plotted onscreen in real time, giving rise to progress curves illustrating the profile of the reaction taking place in the vessel. Result s and Discussion Real time M onitoring of catalysis In order to start every experiment with a set of stable baseline signals for each detected gas, the buffered pre reaction mixture was bubbled for at least 3 minutes either with air (for air equilibrated ex periments) or helium (for deoxygenated or oxygen depleted reaction cond itions). Upon achievement of pre reaction equilibrium, as indicated by a stable flat line ( ~ 0 slope) for each gas signal on the monitor data acquisition begins (t = 0) and the react ion is often initiated with the addition of enzyme after two minutes (Figure 2 4 ) Time at which reaction is initiated by the addition of enzyme may vary depending on the nature of the experiment, but a consistent starting

PAGE 32

32 point is highly preferred in orde r to more conveniently overlay progress curves whenever necessary during data processing. Figure 2 4 Experimental scheme for air equilibrated catalytic experiments. Buffered p re reacti on mixtures containing oxalate substrate are pre equilibrated by the bubbling in of CO 2 scrubbed air. Data acquisition (t = 0) begins when the air inlet is removed. Reaction is initiated upon addition of OxDC enzyme at 2 minutes. (A) (B) Figure 2 5 I on current s (arbitrary scale) in the production of carbon dioxide from (A) 12 C oxalate and (B) 13 C 2 labeled oxalate catalyzed by OxDC in air equilibrated buffer. Dissolved gases were monitored from the ion currents at their respective peak heights: ( red ) 13 CO 2 at m/z 45; ( blue ) CO 2 at m/z 44; and ( green ) O 2 at m/z 32 The so lution contained 50 mM potassium 12 C oxalate (A) and 13 C 2 oxalate (B), 0.2% Triton X 100, 50 mM sodium acetate buffer at pH 4.2 and 25 o C Reaction was initiated at 2 minutes by the addition of ( His 6 tagged) wild type OxD C to a final concentration of in a total reaction volume of 2 mL Examples of real time monitoring of catalysis by MIMS are illustrated in Figure 2 5 Here, OxDC catalysis was initiate d by addition of enzyme at 2 minutes to solution s

PAGE 33

33 containing 50 mM potassium oxalate substrate Real time data are reported onscreen from the instrument as relative ion currents on a logarithmic scale. Logarithmic plotting of detected currents allow for the simultaneous monitoring of small and large changes in the m/z signals in a single frame/screen. Opt imization of sample/reaction conditions is largely dependent on aiming to have the monitored signals stay within the limits of the scale on the screen throughout the catalytic reaction In Figure 2 5A t he progress curve for the accumulation of product CO 2 was observed through the rise of the m/z 44 peak (CO 2 ). A proportional rise in the m/z 45 peak ( 13 CO 2 ) was also observed reflecting the 1% relative natural abundance of 13 C to 12 C in the generation of CO 2 product in solution Alternative ly, catalytic prod uct was observed through the rise in the m/z 45 peak ( 13 CO 2 ) when 50 mM of (99% enriched ) 13 C 2 labeled oxalate was used in a separate experiment (Figure 2 5B ) confirming the assumption that observed rise in CO 2 w as indeed a catalytic product of OxDC from s upplied oxalate substrate. Unlike the previous experiment using unlabeled oxalate, the ratio between m/z 45 and m/z 44 signals in (B) using 99% enriched 13 C 2 oxalate did not amount to 1 or 2%, but an elevated 4%. Although this difference is minimal, the co nsistently observed additional 3% in m/z 44 signal was later confi rmed to be an artifact coming from a small fraction of 13 CO 2 ions being detected by the instrument as 12 CO 2 Because the scanning in the linear quadrupole allows for a sweep of detected ions from high to low m/z, the overlap of frequencies between two adjacent m/z signals result in a small fraction of the m/z 45 ions traveling through the quadrupole to be caught by the detector as part of the detected m/z 44 population of ions This was confi rmed when 99% 13 C labeled potassium bicarbonate was injected in acidic buffer, which gave rise to the same 4%

PAGE 34

34 relative intensity of m/z 44 (vs. m/z 45), instead of the expected 1%. Due to the nature of the frequency sweep (m/z sweep of detection) this phen omenon is limited to adjacent m/z signals and is unidirectional (ie. from higher m/z to lower m/z unit) This would explain is why no artifact signal is observed on m/z 45 when unlabeled oxalate resulting in an abundance of catalytic m/z 44 signal is use d in an experiment In both cases, an apparent consumption of dissolved oxygen was observed in catalysis from the decrease in the (green) m/z 32 signal. In order to confirm this, the experiment utilizing 13 C 2 labeled oxalate was observed with the simultane ous monitoring of an additional gas in solution Dinitrogen, also shown in Figure 2 6 originated from the air equilibration of this sample and is not generat ed or consumed in the catalysis; hence, its peak at m/z 28 i s a control for the stability of the m ethod A very slight decrease in the m/z 28 peak over the course of the experiment (Figure 2 6 ) is a measure of the rate of loss of N 2 from solution by passage across the membrane inlet into the mass spectrometer or into the headspace. This was negligible compared with changes in 13 CO 2 levels. A small component of total catalysis by OxDC is an oxidase that consumes oxygen and yields two molecules of CO 2 in each catalytic cycle ( Figure 1 1 B ) Consumption of oxygen in Figure 2 6 is observed by the rate of dec rease in the m/z 32 peak which is greater than the rate of decrease in the control peak for m/z 28. A further control was performed in a separate experiment in which dioxygen was introduced into the reaction buffer containing oxalate but in the absence of OxDC. The observed rate of signal decay for m/z 32 was negligible From the data of Figure 2 6 and similar repeated experiments, the ratio between the calibrated rate s of decrease in m/z 32

PAGE 35

35 (apparent O 2 consumption) and the rate of increase in m/z 45 (cat alytic 13 CO 2 ) amounted to 0.3 0.5%. This led to the estimate that approximately 0. 3 % to 0.5% of the overall rate of catalytic degradation of oxalate is due to the oxidase pathway of catalysis by OxDC rather than the decarboxylase pathway which is consis tent with literat ure v alues ( 7, 37 ) Hence the MIMS method has the capability to measure the oxidase function of OxDC in the same experiment as the decarboxylase activity. Figure 2 6. Ion current in the production of 13 CO 2 from 13 C 2 oxalate catalyzed by OxDC Dissolved gases were monitored from the ion currents at their respective peak heights: ( red ) 13 CO 2 at m/z 45; ( green ) O 2 at m/z 32 ; and ( cyan ) N 2 at m/z 28. The solution contained 50 mM potassium 13 C 2 oxalate, 0.2% Triton X 100, 50 mM sodium acetate buffer at pH 4.2 and 25 o C Reaction was initiated at 2 minutes by the addition of ( His 6 tagged) wild type OxD C to a final concentration of in a total reaction volume of 2 mL Reprinted from ( 36 ) with permission by Elsevier. Reproducibility In order to assess the reproducibility of observe d catalysis using MIMS data from three separate experiments of identical conditions to those described in Figure 2 6 were compared (Figure 2 7 ). The experiments in re d and blue were separated by 20 minutes.

PAGE 36

36 The experiment in black was performed on a separate day. The standard deviation in the initial rates (5% product conversion ) in these three measurements was 6%. Figure 2 7 Superimposed ion currents (arbitrary sca le) of three separate experiments involving the production of 13 CO 2 from 13 C 2 oxalate catalyzed by OxDC. Dissolved gases were monitored from the ion currents at their respective peak heights for 13 CO 2 at m/z 45 and O 2 at m/z 32. The solution contained 50 m M potassium 13 C 2 oxalate, 0.2% Triton X 100, 50 mM sodium acetate buffer at pH 4.2 and 25 o C. Reactions were initiated at time 0 by the addition of (polyhistidine tagged) wild a total reaction volume of 2 mL. Reprinted from Supplementary Material of ( 36 ) with permission by Els evier Response time The response time of the apparatus was measured by rapid injection into the reaction vessel of a solution containing CO 2 and measuring the time to reach a plateau of ion current at m/z 44. The final concentration of CO 2 was approximate ly 2 mM and the time to reach plateau was adequately fit to a first order process with a half time of 4 seconds. Buffer conditions: Air equilibrated vs. O 2 depleted (He bubbled) A ir equilibrated buffers were selected in this chapter to best parallel the ex perimental conditions of the traditio nal FDH coupled endpoint assay. H owever it was

PAGE 37

37 observed that catalysis was not significantly different when buffers were considerably depleted of dissolved dioxy gen. In these experiments buffers were alternatively bubb led in with helium gas minutes prior to catalysis as described in Figure 2 8 Figure 2 8 Experimental scheme for oxygen depleted catalytic experiments. Buffered pre reaction mixtures containing oxalate substrate are pre equilibrated by the bubbling in o f helium gas Data acquisition (t = 0) begins when helium inlet is removed. A gentle sweep of helium is placed on the solution surface to prevent air from the headspace to re enter the solution. Reaction is initiated upon addition of air equilibrated OxDC enzyme at 2 minutes. (A) (B) Figure 2 9 I on current s in the production of carbon dioxide from (A) 12 C and (B) 13 C 2 labeled oxalate potassium oxalate catalyzed by OxDC in deoxygenated buffer. Dissolved gases were monitored from the ion currents at th eir respective peak heights: ( red ) 13 CO 2 at m/z 45; and ( green ) O 2 at m/z 32 The solution contained 50 mM potassium 12 C oxalate (A) and 13 C 2 oxalate (B), 0.2% Triton X 100, 50 mM sodium acetate buffer at pH 4.2 and 25 o C Reaction was initiated at 2 minute s by the addition of ( His 6 tagged) wild type OxD C to a final concentration of in a total reaction volume of 2 mL

PAGE 38

38 Addition of 5 20 Ls of air equilibrated enzyme stocks (0.6 2.5 M delivered oxygen) still resulted in observed decarboxylase activity albeit without any drop in the m/z 32 signal earlier associated with oxyge n consuming oxidase activity. Thus, O 2 depleted buffer conditions were subsequently used (in Chapters 3 and 4) especially in experiments involving agents that are easily oxidized; and catalytic measurements requiring selectivity for decarboxylase activity. Background CO 2 and Isotope labeled substrate ( 13 C 2 oxalate) Because catalys is is i nitiated b y addition of air equil ibrated enzyme stocks, carryover of CO 2 from enzyme aliquots often result in a small non catalytic rise in detected m/z 44. Magnitude of thi s non catalytic signal is recorded from a separate control experiment in the absence of substrate, where the maximum ion current from this CO 2 is noted together with the time the signal reaches its maximum (Figure 2 10) I n most cases when con siderable catalytic a ctivity is observed, effects of this background CO 2 on observed initial rates are negligible (Figure 2 10B) Nonetheless, this background signal is either subtracted at the end of the experiment, or neglected by measuring initial rates from a time coordinate when this artifact has completely passed through the detector In cases where low catalytic rates are anticipated, or measured in the presence of elevated amounts of dissolved CO 2 the use of 13 C 2 isotope labeled oxalate is often pr eferred. This allows the convenient detection of catalytic carbon dioxide by the alternative monitoring of increasing m/z 45 ( 13 CO 2 ), which can only be observed in solution by the presence of OxDC and unaffected by carried over CO 2 from the enzyme aliquot

PAGE 39

39 (A) (B) Figure 2 10 Overlay of m/z 44 ion currents illustrating background (red) CO 2 detected from the air equilibrated OxDC enzyme aliquot in the absence of substrate. (Blue) is the ion current from the catalysis of 10 mM oxalate by the same amount of OxDC enzyme. (A) presents the overlay of m/z 44 ion currents on a logarithmic scale. (B) is the same data showing the first minute of detected m/z 44 ion currents on a linear scale to illustrate differences in magnitude o Phenylenediamine (o PDA) Emilian i et al d ocumented the favorable effect of reducing substances such as o phenylenediamine (o PDA) on catalysis by OxDC from A. niger ( 37 ) O PDA had been a constant component of the endpoint assay reaction mix, presumably contribu ting to the stability of the enzyme for adequate detection of its activity through out the coupled assay protocol. For the MIMS approach, o PDA was no longer included in the buffer mix for two reasons: i) n o significant difference was observed in MIMS detec ted catalysis in the ab sence of it; and ii) unnecessary reactive buffer comp onents may contribute to signal artifacts from non enzymatic side reactions. Calibration In order to convert raw ion currents to millimolar (mM) or micromolar ( M) concentrations o f detected gases, t he membrane inlet mass spectrometer was calibrated by rapid injection into the reaction vessel of solutions of known CO 2

PAGE 40

40 concentration. The most accurate procedure was to prepare solutions of K 2 CO 3 at pH 10.2 and inject known volumes int o the reaction vessel containing a concentrated solution of acetic acid (pH ~ 2) At this pH, 99.9% of all carbonate species exist as CO 2 The ion current at m/z 44 was recorded when it reached a maximum. A plot of ion current versus CO 2 concentration was linear (Figure 2 11 ). We did not test the upper or lower limits of detectability of CO 2 since these were not pertinent to the measurement of catalytic activity of OxDC The instrument was similarly calibrated for O 2 by recording the average ion currents a t m/z 32 in solutions of different O 2 concentration prepared by dilution of air saturated reaction buffer at 25 o C ([O 2 ( 38 ) The r esulting plot of ion current versus O 2 concentration was linear. The baseline signal corr esponding to 2 was verified using degassed reaction buffer containing 1 mg glucose oxidase which was deoxygenated by the addition of 7 mM gluco se ( 7, 39 ) Figure 2 11. Ion current (arbitrary scale) at m/z 4 4 using the membrane inlet mass spectrometer observed using solutions of known CO 2 content. Solutions containing CO 2 were prepared from step additions resulting in 0.25 mM increments of K 2 CO 3 into concentrated acetic acid (pH 2.0) and 25 o C. Peak heights we re recorded when equilibrium was reached. Shown are the averages of three repetitions with relative standard deviations of 3% at 4.8 mM CO 2 8% at 3.0 mM CO 2 and 10% at 0.4 mM CO 2 The solid line is a least squares fit with a correlation coefficient of 0. 999. Reprinted from ( 36 ) with permission.

PAGE 41

41 Derived slopes from these linear fitted calibration plots are used as factors to convert raw ion currents to millimolar or micromolar amounts of CO 2 or O 2 Whenever necessary, ion currents of o ther gases may similarly be calibrated However, because different gases may interact an d pass through the membrane differently, separate calibration experiments for those gases are required. Derived slopes are often good throughout a batch of experiments when the sensitivity (photomultiplier) settings and installed membrane remained unchange d. New calibration data is often required in situations when a new sensitivity setting is used, or when a new membrane (or any component of the instrument) is installed, as such would change the response of the instrument for the subsequent sets of experim ents. Figure 2 12. Ion current (arbitrary scale) at m/z 32 using the membrane inlet mass spectrometer observed using solutions of calculated O 2 content. Amounts of dissolved O 2 in solution were varied by diluting deoxygenated buffer with known volumes of air equilibrated water to a final volume of 2 mL. Air equilibrated buffer at 25 o C was assigned a concentration of 256 M according to the solubility of O 2 in water at 25 o C ( 38 ) .Corresponding m /z 32 signal for 0 M O 2 was taken from buffers deoxygenated by the prior bubbling in of helium gas. Shown are the averages of duplicate runs with standard deviations of 10% at 256 M O 2 and 8% at 61 M O 2 The solid line is a least squares fit with a correlation coefficient of 0.999.

PAGE 42

42 Data Analysis : Michaelis Menten Kinetics Steady s tate Briggs and Haldane introduced the assumption of steady state as the point in enzym atic catalysis where reaction intermediates reach a constant level, resulting in a stable unchanged react ion rate as a function of time. The Michaelis Menten equation expresses this concept through a hyperbolic function relating reaction rate (v) with subs trate concentration [S], where V max is the hypothetical maximum rate achieved, which is no longer affected by increasing substrate concentration. The turnover number k cat is related to V max in Eq n 2 2 where [E] is the total enzyme concentration. Michaelis constant (K m ) is an experimental term describing the concentration of substrate at half the maximum rate (V max ) ( 40 ) (2 1) (2 2) The hyperbolic shape of this function is a result of two possible scenarios covered by this equation across various levels of substrate concentration [S]. At very low substrate co ncentrations where [S] << K m v can be described by Eqn 2 3. ( 40 ) (2 3) As substrat e concentrations increase where [S] >> K m the quantity K m becomes negligible compared to [S] and thus v alternatively becomes determined by V max which ideally becomes unaffected by further increases in [S]. In these conditions the enzyme ( 40 )

PAGE 43

43 (2 4) Because these two scenarios are distinguished by the experimental quantity K m catalytic rates (v) at varying substrate concentration [S] are measured with the aim for deriving kinetic const ants through the fitting of [S] vs. v plotted data to the Michaelis Menten equation ( E q n 2 1). In order to experimentally accomplish this, earlier assumptions are maintained in the experiment. Because steady state implies overwhelming amounts of substrate versus enzyme, en zyme concentrations are ideally at least three orders of magnitude less than total substrate concentration ( 40, 41 ) Under these conditions, when total enzyme concentration is occupied with bound substrate, the change in remaining [S] is negligible. Furthermore, the highest at tainable rate at any given substrate concentration would conceptually be at the early stage of catalysis when the putative enzyme substrate complex is formed and changes on [S] is still negligible. This rate is often hich often translates to ~5% 10% product conversion ( 41 ) and is thus the main quantity of inter est whenever a catalytic ra te is reported. Initial velocities Initial velocities in catalytic experiments are often manifested through the early linear increase in the progress curve of product formation. These velocities are maximum rates achieved in those conditions before the pro gress curve plateaus either by significant depletion of available substrate to be converted or by reaching the limit of enzymatic turnover. Because the progress curves for CO 2 formation through MIMS are preceded by a lag time due to the response time of th e instrument, linear points

PAGE 44

44 immediately after this lag time are sampled and fitted to a linear function whose slope is representative of the catalytic rate for a given substrate concentration (Figure 2 13) Sources of e rror Sources of experimental error in this method may include pipetting and the heterogeneity of stock solutions used throughout each set of measurements. These sources of error can be minimized by the use of the same pipets and stock solutions throughout each experiment and by having a stand ard way of mixing and delivering the buffer components with care into the reaction vessel. (A) (B) Figure 2 13. Example of deriving initial velocity from a progress curve of CO 2 formation. (A ) P rogress curve of calibrated ion currents showing 13 CO 2 form ation from the catalysis of 30 mM 13 C 2 labeled oxalate by 1.44 M OxDC at pH 4.2. Inset are linear fits of two sets of sampled points bracketed in red and green f or deriving the initial rate Embold ened linear slopes 5% error in the derived initial rate, which is plotted on a separate plot ( B ) of reaction rate vs. oxalate concentration. Unless otherwise stated, each point represents a single measurement from the fitting of at least 10 data points in a single progress c urve. R eported standard errors are from the variations of the slopes derived from the single progress curve Because of the reproducibility of observed ion currents the major source of errors is associated with how the data is fitted and processed. These include the fitting of calibration points to derive a factor to convert i on currents to mM concentrations, and

PAGE 45

45 further fitting to derive reaction velocities from sigmoidal progress curves (Figure 2 13) whic h may give rise to an error of 1 % 8 % in the der ived slopes (initial velocities) Hence, errors presented in the catalytic rates are errors inherent to deriving the reaction velocities from the progress curves I n the following sections on Michaelis Menten kinetics, single measurements of catalytic rate s were ma de over more points of oxalate concentration to construct the Michaelis Menten curve of this enzyme, in stead of doing replicate runs over fewer substrate concentrations Data are presented in the following sections. Kinetic constants (MIMS vs FDH) Figure 2 14 Accumulation of 13 CO 2 in solution resulting from the catalysis by OxDC.. Initial concentrations of 13 C 2 labeled (99%) oxalate were as follows: red, 0.5 mM; dark blue, 1 mM; green, 2 mM; black, 4 mM; purple, 8 mM; cyan, 16 mM; yellow, 30 mM; orange, 50 mM; light blue, 80 mM. Each curve for different concentrations of oxalate represents a single experiment. Other components of the sol utions were as described in Figure 2 5 Reprinted from ( 36 ) with permission by Elsevier

PAGE 46

46 Figure 2 15. Initial rates of the appearance of 13 CO 2 in solutions resulting from cataly sis of the decarboxylation of oxalate by OxDC. Each data point represents a single measurement of initial velocity from the data of Figure 2 14. The solid line is a fit to the Michaelis Menten equation (Eqn 2 1), with constants for catalysis given in Table 2 1 Reprinted from ( 36 ) with permission by Elsevier. Oxalate conta ining two 13 C labels (99% 13 C) was used as substrate to distinguish catalytically generated 13 CO 2 (m/z 45) from pre existing 12 CO 2 (m/z 44) in reaction samples. Progress curves were measured to show catalytically generated 13 CO 2 at various initial concentr ations of oxalate ; the initial segments of such curves are shown in Figure 2 14 The slow phase in the beginning 8 12 sec is the response time. The initial velocities of the catalytic production of CO 2 were determined from the linear slopes of Figure 2 1 4 at times 5% to 10% of approximate product conversion. The rate of the uncatalyzed reaction is negligible as is the loss of CO 2 into the instrument and headspace. The initial rates of catalysis were adequately fit to a simple Michaelis Menten curve ( Figur e 2 15) with catalytic constants given in Table 2 1 These constants

PAGE 47

47 are in reasonable agreement with those determined by coupled assay using NAD requiring formate dehydrogenase ( Table 2 1 ). The Michaelis Menten plot for the formate dehydrogenase assay is shown in Figure 2 16. From a concomitant decrease in the m/z 32 peak, such as shown in Figure 2 2 the measured rate of O 2 consumption which was determined to be 0.3% to 0.5% of the rate of overall catalysis by OxDC at saturating substrate (50 mM oxalate) and air equilibration, wa s due to the inherent catalysis of the oxidase reaction ( Figure 1 1B ). Under similar conditions Tanner et al estimated this value to be 0.2% ( 7 ) This is too small a rate to affect significantly the constants for the decarboxylase activity given in Table 2 1 MIMS measurement of product CO 2 in cat alysis by OxDC from B. subtilis has obtained steady state constants in reasonable agreement with those by an endpoint assay using formate dehydrogenase ( Table 2 1 ). The values of k cat and k cat /K m were somewhat larger when measured by MIMS, perhaps related to the advantages of MIMS in being able to provide a sensitive, continuous, and real time measure of CO 2 in solution. Table 2 1. Steady state constants for decarboxylation of oxalate catalyzed by polyhistidine tagged OxDC measured by MIMS and by formate de hydrgenase coupled assay. Uncertainties are standard errors in the fit to the Michaelis Menten expression (Equation 2 1) Reprinted from ( 36 ) with permission by Elsevier Assay K m (mM) k cat (s 1 ) k cat /K m (mM 1 s 1 ) MIMS 4.0 0.5 71 2 1 8 1 FDH coupled Endpoint Assay 3.9 0.4 44 1 11 1

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48 Figure 2 16 Initial ra tes of catalysis by polyhistidine tagged B. subtilis oxalate decarboxylase determined by the formate dehydrogenase coupled endpoint assay descri bed in the Experimental section. One minute experiments for the rate of formate production (mM/min) at each oxal ate concentration wer e done in triplicate and fitted to the Michaelis Menten expression (Eq n 2 1) resulting in an R 2 of 0.92334. The solid line is a fit to the Michaelis Menten expression with d erived steady state constants given in Table 2 1 Reprinted f rom Supplementary Material ( 36 ) with permission by Elsevier Experim ental Section Materials Protein concentrations were determined using the Coomassie Protein Plus Kit (Pierce, Rockford, IL) with calibration curves generated utilizing bovine serum albumin as standard. DNA sequencing services were done by the core facility of the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. Solutions of carbon 13 oxalate ( 13 C 2 labeled oxalic acid; 99% 13 C; Cambridge Isotope Laboratories Andover, MA) were prepared by adjusting pH to 4.2 with minim um amounts of potassium hydroxide

PAGE 49

49 His 6 tagged Oxalate Decarboxylase Expression and Purification The OxDC:pET 32a plasmid construct for the polyhistidine tagged wild type B. subtilis OxDC was acquired from the research group of Dr. Stephen Bornemann of the John Innes Centre in Norwich, UK. This plasmid was cloned into BL 21/DE3 E. coli cells in which the polyhistidine tagged wild type OxDC was expressed and later purified using previously established methods ( 14, 15 ) except that grown cells were lysed via sonication. Pooled elution fractions from Ni NTA agarose (Quiagen) affinity chromatography were eluted through a 100 mL G 25 Sephadex Desalting column equilibrated with 50 mM Tris Cl (pH 8.5) and 0.5 M NaCl storage buffer. Enzyme samples were treated with Chelex 100 resin (BioRad) for at least 1 hour with gentle swirling to remove trace metals. Final samples were buffer exchanged into Chelex treated storage buffer and further concentrated to within 6 10 mg/mL usi ng YM 30 Centriprep concentrators (Millipore). Purity was assessed from the 44 kDa protein band on a 12% resolving SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, from protein fractions taken at different stages of the purificatio n. Metal incorporation of purified protein (2.25 mgs) was quantified in 1% nitric acid protein solutions ( 18 ) by the ICP MS (inductively coupled plasma mass spectrometry) ( 42 ) services of the University of Wisconsin Soil and Plant Analysis Laboratory. Our samples of OxDC contained 1.4 Mn/monomer Each monomer cont ains two manganese binding sites, one of which is catalytic ( 1, 3, 43 ) We do not know the distribution of metal ions between these two sites. In the kinetic results reported here, the concentration of enzyme has be en set equal to the protein content of solution.

PAGE 50

50 Formate Dehydrogenase (FDH) Coupled Activity Assay OxDC activity was measured through an endpoint assay coupled with NAD + requiring formate dehydrogenase enzyme ( 14, 1 8 ) Assay reaction mixtures were composed of 50 mM sodium acetate buffered at pH 4.2, 0.2% Triton X 100, 0.5 mM o phenylenediamine, and 4 125 mM potassium oxalate. Reactions were initiated with the addition of a t ambient temperature of 23 25 o C. The reaction was quenched after a predetermined formate product wa s quantified by a coupled assay reaction using 50 mM potassium phosphate buffered at pH 7.8, 1.4 mM NAD + and 0.4 1.0 U/mg of formate dehydrogenase at a final volume of 1 mL. The amount of NADH was measured by absorbance of assay mixtures at 340 nm a fter o vernight incubation at 37 o C. Corresponding production of formate was quantified against a calibration curve generated from the relative absorbances A 340 of pre quenched OxDC assay mixtures containing known amounts of formate ( 18 ) Membrane Inlet Mass Spectrometry (MIMS) The inlet probe to the mass spectrometer (Figure 2 1) comprised a 1 cm length of silicon rubber tube (1.5 mm i.d. and 2.0 mm o.d., Silastic a Dow Corning product), which was sealed by a gla ss bead at one end and interfaced to an Extrel EXM 200 quadrupole mass spectrometer ( 35 ) The inlet probe w as immersed in a 2 mL reaction solution contained in a gas tight quartz cuvette (1 cm pathlength). The reaction vessel was jacketed at a temperature of 25 o C, and sealed with injection septa and Teflon screws for the introduction of samples and inert gas. T his apparatus is previously described ( 44 )

PAGE 51

51 Experiments were initiated by the a ddition of enzyme or substrate, and masses were recorded continuously using an Extrel EXM 200 mass spectrometer at an electron impact ionization of 70 eV using an emission current close to 1 mA. Source pressures were approximately 1 x 10 6 torr. The reacti on vessel was washed between experiments with a solution of 2.5 M KOH and 2.5 M EDTA and thoroughly rinsed with deionized water in order to prevent any carry over of residual enzyme or unreacted material. Reaction Mixtures R eaction solutions for MIMS meas urement contained the same components (except o phenylenediamine) as those described in the FDH Coupled Activity Assay section, but scaled up to a final volume of 2 mLs In order to more carefully assay initial rates of enzymatic CO 2 production and from de tected non enzymatic CO 2 in solution, e ither unlabeled or 13 C 2 labeled oxalate (99%) was used as substrate A trace amount of Antifoam A was added to prevent any loss of solution due to frothing when pre reaction buffers were equilibrated by the bubbling o f either air or helium gas (deoxygenated reaction conditions). Depending on the desired buffer conditions, pre reaction buffers were either air equilibrated or deoxygenated (helium gas bubbling) until MIMS analysis showed baseline levels. To minimize any interfering signals from detected gases entering the reaction solution, a slow but continuous flow of air or helium gas was maintained on the headspace throughout the acquisition of data. R eactions were initiated by the addition of the required wild type, His 6 tagged OxDC so that the final enzyme concentrations which was comparable to the amount of enzyme earlier used in the kinetic experiments employing the FDH endpoint assay. In later experiments (Chapter 4) enzyme aliquots used to initiate c atalytic react ion were reduced to about 30 40% of the

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52 original enzyme aliquot described above Although the original recipe worked just fine, reducing the amount of wild type enzyme not only extended the stock of purified enzyme, but also reduced the rate of catalytic generation of CO 2 enough for the instrument to report more points in the linear increase of the catalytic progress curves, where initial velocities were best sampled.

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53 CHAPTER 3 NITRIC OXIDE INHIBITION OF CATALY SIS BY OXDC Introduction I t is presently believed that decarboxylation proceeds from an oxalate radical anion intermediate, which is generated in a proton coupled electron transfer step mediated by the Mn(II) ion ( 23 ) In addition, the reported depen dence of the enzyme catalyzed chemistry on the presence of dioxygen ( 7 ) has led to the proposal that formation of the oxalate radical anion takes place via a Michaelis complex in which both oxalate and dioxygen are boun d to the catalytically active manganese center ( 10, 18, 22 ) Direct support for this hypothesis has yet to be obtained, however, and there is an absence of an unambiguous chemical precedent for the interaction of di oxygen with high spin Mn(II) inorganic complexes. Efforts to observe the proposed interaction of dioxygen with the metal in OxDC have also been complicated by the fact that the two Mn(II) centers in the enzyme exhibit similar EPR properties ( 16, 17 ) Nitric Oxide as Dioxygen Mimic In an effort to assess the existence of the putative di oxygen binding site in OxDC, the interaction of nitric oxide (NO) with the enzyme was examined using the new continuous assay based on membrane inlet mass spectrometry (MIMS) mentioned in C hapter 2 ( 44, 45 ) Nitric oxide is similar to dioxygen in that it is a linear uncharged molecule which can also enter the membrane inlet of the instrument and b e simultaneously detected with other gases in the solution throughout catalysis. Nitric Oxide from NONOates Nitric Oxide (NO ) was generated in situ with the use of NONOates which are stable organic compounds at basic pH 8.5. At pH 7.0 these compounds deco mpose,

PAGE 54

54 generating known amou nts of nitric oxide in solution ( 46 48 ) In acidic conditions ( pH 4.2 ) the s e NO generating reaction s are instantaneous making NONOates a non invasive and efficient source of NO for the Ox DC catalytic reaction mixtures in this chapter Fig ure 3 1. Structures of MAHMA / DEA NONOates at pH 8.5, and their respective reaction byproducts at pH 7. ( 46 48 ) Because nitric oxide rapidly oxidizes in the presence of oxygen, especially in acidic conditions ( 49 ) experiments in this chapter were conducted under deoxygenated buffer conditions (ie. equilibrated by the bubbling of helium). In order to test for the effects of nitric oxide on catalysis by OxDC, the overall pre re action scheme had to be slightly modified ( Figure 3 2 ). In the modified scheme, reaction buffers were deoxygenated and equilibrated as previously described prior to data collection (t = 0 mins). In contrast to previous runs, the reaction buffer containing substrate was then injected with a known amount of NONOate in order to populate the reaction mixture with known amounts of nitric oxide ( Figure 3 2 blue path) prior to initiating the reaction by the addition of OxDC enzyme.

PAGE 55

55 Figure 3 2 Modified experimen tal scheme for testing the effects of NO on catalysis by OxDC. Reactions were performed under deoxygenated conditions (orange path) similar to previously described wild type experiments in Chapter 2. Modified scheme is illustrated by the divergence in the scheme (blue path), where the reaction buffer is first populated with NO, prior to the initiation of catalysis by the addition of OxDC enzyme. Data Analysis: Inhibition The hyperbo lic Michaelis Menten function ( E qn 2 1) can be linearized by taking its inv erse ( E qn 3 1 ) and plotting catalytic rates (v) at varying substrate concentration [S] on a double reciprocal (Lineweaver Burk) plot 1/[S] vs. 1/v ( 50 ) This approach facilitates a good preliminary estimate of the kinetic parameters of the catalytic experiment through a least squares fitting of the data points to these reciprocal equations. (3 1) Although lesser points are required to derive a good fit to a linear function than to a highly resolved hyperbolic curve, a disadvantage of the reciprocal approach is the inherent error on derived catalytic constants contributed by the lower catal ytic rates at low substrate concentrations (farthest points from the axes). Because these lower catalytic rates are experimentally harder to measure and thus prone to the most error,

PAGE 56

56 variances in these measurements graphically have a considerable effect on the slope (K m /V max ) and extrapolated y intercept (V max ) of the fitted linear equation. This effect becomes a greater concern in inhibition experiments where diminished catalytic rates (compared to wild type controls) are expected and when estimates for th e mode of inhibition are accomplished through global fits. Modes of inhibition presented later in the chapter and in Chapter 4 were derived by globally fitting sets of catalytic rates at varying inhibitor and substrate concentration to kinetic models of si mple competitive, uncompetitive, and mixed/noncompetitive. The program incorporates a v 4 weighting algorithm in this process, which will later be described. Different kinetic models have qualitative implications on the inhibitor enzyme interactions and can be expressed in modified rate equations on which the global fitting program is based. Theoretical effects of inhibitors are expressed by the function which relates inhibitor concentration [I] with its corresponding dissociation constant K i By definitio n, K i refers to the concentration of inhibitor required to occupy half the total available enzyme at steady state conditions. distinguishes itself from by the form of the enzyme it targets in catalysis, which will be la ter discussed. (3 2) Although much of these concepts will be revisited in Chapters 3 and 4, it would be useful at this point to describe the main implicatons of the different modes of inhibition and how they relate to the kine tic parameters derived from the data.

PAGE 57

57 Michaelis Menten kinetics is grounded on the basic enzymatic scheme where free enzyme [E] interacts with substrate [S] to form a hypothetical form/complex [ES]. This form undergoes catalysis to form product [P] which i s then released, regenerating the free form enzyme [E] for another catalytic cycle ( 40 ) : Figure 3 3 Fundamental enzymatic reaction model. The model is limited by the fact that it views the reaction from the perspective of reactants and products and thus gives no clear indication of mechanism, or individual steps within the scheme whi ch may very well exist. Nonetheless inhibitors in theory, interfere with catalysis by targeting different hypothetical forms of the enzyme in the catalytic scheme, depending on their mode of inhibition ( 40, 41 ) Competitive inhibitors are species which target the free form of the enzyme [E] (Figure 3 4) to bind to and undergo catalysis. Although many competitive inhibitors are structural analogues of the substrate, a competitive inhibitor may not alway s be assumed to bind to the exact same binding site of the substrate on the enzyme. Figure 3 4 Enzymatic s cheme for c ompetitive i nhibition. Because they interfere with the interaction of substrate on the enzyme, more sub strate is essentially required to achieve V max K m is essentially affected even though V max ideally remains unchanged. From the linearized Michaelis Menten equation earlier

PAGE 58

58 derived, the modified equation describing a competitive mode of inhibition is expre ssed m term. ; where (3 3) Uncompetitive inhibitors by contrast, inhibit the form of the enzyme when substrate is present (Figure 3 5) This means that it targets the form of the en zyme where substrate is either bound, or any downstream form prior to product released, where the free form of the enzyme is regenerated. Figure 3 5 Enzymatic s cheme for u ncompetitive i nhibition In terms of kinetic par ameters, uncompetitive inhibitors affect both K m and V max in a proportional way. A slight distinction in the similarly defined function and K i i ndicates a distinct form of the enzyme [ES] it targets from the free form [E] whe re the presence of [S] is i rrelevant. ; where (3 4) Mixed / Noncompetitive inhibitors are those which target both forms of the enzyme, indicating that they have the ability to inhibit whether substrate is present or not (Figure 3 6) Mixed and noncompetitive modes of inhibition share this common feature and differ only on whether the inhibitor has equal affinity for both forms of the enzyme, where

PAGE 59

59 the tw o forms it can target, ie. Ki For simplicity, mixed modes of inhibition will be considered similar to noncompetitive, which is distinct from previously described competitive and uncompetitive mode l s. Because noncompetitive/mixed inhibitors i nhibit regardless of the presence of the substrate, it is implied that these species interact with the enzyme at a location on the enzyme other than the substrate binding site. Figure 3 6 Enzymatic scheme for noncompetit ive/mixed inhibition The rate equation associated with this mode of inhibition (Eqn 3 5) is described as a combination of the two previously described equations, where the respective effects of the two illustrated K i s (Figure 3 6) are reflected by the cor responding and denotations. These effects simply convey the unique feature of this mode, where the inhibitor targets both [E] and [ES] forms of the enzyme as indicated in Figure 3 6. (3 5) G iven these different models of inhibition t he fitting program uses a series of matrix elimination functions to find the kinetic parameters which best fit the spread of initial velocities (discussed in Chapter 2) measured at varying inhibitor and substrate concentrations. Kinetic parame ters derived for each of the three possible modes of inhibition are assessed in this process with the best fit often indicated by an R 2 correlation value of no less than 0.9. Bad fits often reveal themselves with correlation values of 0.8 or less.

PAGE 60

60 In order to compensate for the error described from the linearization of the Michaelis Menten equation, weighting factor (w i ) was incorporated in the fitting program used on the catalytic rates (v i ) in ou r inhibition experiments (Eqn 3 6) (3 6) This factor essentially biases the fitting towards putting more weight on the higher velocities measured, where errors are presumably not as high. When the variance of measured velocities are assumed to be constant, the weighting factor essent ially becomes v 4 ( 51 ) hence the name of the algorithm. This assumption holds true in many cases ( 51, 52 ) which is the case in each of our mode of inhibition experiments. Furthermore, this assumption of constant variance assumes that the range of measured velocities across all substrate concentrations is no more than a factor of 5. In cases when this does not hold true and experimental velocities for example, range by a power of 10 or more, the assumption of constant vari ance no longer applies and results in the exclusion of the lower velocities in the v4 weighted fitting process ( 50 ) This may explain why graphical representations of the fitted parameters of our data give a much poorer fit to the data sets of highest inhibitor concentrations and low substrate concentration. In order to illustrate the results of the global fitting of the data, coordinates are manually calculated from the constants and model equation derived by the fitting software. Specific modes of inhibition are indicated by distinct patterns of lines resulting from the fitting of catalytic at varying inhibitor and s ubstrate concentration ( Table 3 2 ) Linearized catalytic rates are often plotted on a double reciprocal (Lineweave r Burke) plot of 1/v vs. 1/[S]. However an alternative known as the Hanes plot, is preferred on

PAGE 61

61 account that the plot minimizes the effect of the errors from lower catalytic rates at low substrate concentrations ( 41 ) Hanes plots by contrast, plot substrate concentra tion [S] against the reciprocal of the corresponding initial velocity multiplied by the substrate concentration ([S]/v) Derived kinetic constants are illustrated through distinct graphical features o f each plot as summarized in Table 3 1 ( 40, 41, 53 ) Table 3 1 Table of comparison between graphical features of Lineweaver Burke and Hanes plots. Graphical Features Lineweaver Burke Plot Hanes Plot Linear rate equation Slope of the line y intercept x intercept K m Table 3 2 Modes of Inhibition represented by Lineweaver Burke and Hanes Plots. Linear patterns in each plot resulting from the linear fits of initial reaction velocities at varying substrate and inhibitor concentrations Mode of Inhibition Lineweaver Burke Plot Hanes Plot Competitive Inhibition Intersecting lines a t y axis Parallel Lines Uncompetitive Inhibition Parallel Lines Lines converging at y axis Noncompetitive Inhibition Lines converging at negative x axis Lines converging at negative x axis Mixed Inhibition Lines intersecting at a point in quadrant I ( ie. where x < 0 and y >0) Lines intersecting at a point in quadrant I (ie. where x < 0 and y >0) Results and Discussion Reversible Inhibition of NO Figure 3 7 shows the decrease in initial activity of the enzyme by approximately two orders of magnitude i n the presence of NO, as indicated by the reduction in the

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62 observed ion current for 13 CO 2 (m/z = 45) Here, an assay solution containing 50 mM 13 C 2 oxalate in 50 mM acetate buffer, pH 4.2, was degassed with He to deplete dioxygen. Air in the head space abo ve the reaction mixture was then removed with He for 2 min prior to the a d dition of 25 M MAHMA NONOate ( 46, 47 ) Under the acidic conditions, this reagent rapidly decomposed to yield 50 M NO ( F igure 3 7 ). OxDC (1. 4 mM) was then added to the solution at 4 min to initiate the decarboxylation reaction (F igure 3 7 ) Under these conditions, the ion associated with NO (m/z = 30) was also detected. Figure 3 7. Effect of NO on catalysis by C terminally His 6 tagged OxDC. Time course of changes in ion currents (arbitrary units), determined using MIMS for 13 CO 2 ( red ), NO ( blue ) and O 2 ( green ). At 2 minutes, 25 M MAHMA NONOate was injected into deoxygenated buffer containing 50 mM 13 C 2 oxalate, 0.2% Triton X and 50 mM Aceta te pH 4.2. Catalysis was initiated 2 minutes later by the addition of 1.44 M His 6 tagged OxDC. CO 2 free air was bubbled in for 30 seconds at 12 minutes. (Inset) The same set of data plotted on a linear axis to emphasize the magnitude of the ion currents f or 13 CO 2 ( red ) and NO (blue). Reprinted from ( 54 ) with permission by the Royal Society of C hemistry Even in the presence of NO, the enzyme was capable of catalyzing the formation of a small amount of 13 CO 2 immediately on addition, which likely results from the

PAGE 63

63 presence of the bound dioxygen on the enzyme added to the reaction mixture. The time dependent decrease in the ion current for both these species over the next 8 min was due at least in part, to the loss of CO 2 and NO from the reaction solution through the membrane inlet into the mass spectrometer. At 12 min, air was re introduced, resulti ng in the restoration of full OxDC catalytic activity, albeit after a time lag of approximately 5 min ( Figure 3 7 ). Upon introduction of air by bubbling, some CO 2 was lost from solution, giving rise to a decrease in the ion current of the m/z 45 peak. Fi gure 3 8 Effect of N 2 on catalytic inhibition by NO. Same r eaction conditions as in F igure 3 7 except that reaction was initiated with M NONOate treated enzyme stock and instead of air N 2 (cyan) was bubbled in at t = 24 minutes, followed by CO 2 free air 20 minutes later. To confirm that recovered activity was not simply due to the removal of NO from solution as a consequence of bubbling in air, a similar experiment was conducted where NO was deliberately removed from the inhibited reaction mix by the bub bling of nitrogen

PAGE 64

64 gas instead of air ( Fig ure 3 8 ). The fact that catalytic CO 2 was only recovered after the later bubbling in of air confirmed that inhibition was indeed reversed by dioxygen. Figure 3 9. MIMS experiment showing the effect of adding MAHMA NONOate to OxDC during catalytic turnover. Pre reaction buffers were deoxygenated as described in Figure 3 7. After 2 min, His 6 tagged, wild type OxDC (1.4 was added to initiate reaction. CO 2 generation took place immediately (red). After an additional 20 seconds,a solution of 50 M of MAHMA NONOate was injected into the reaction mixture and NO formation was observed (blue). The dioxygen concentration und er these conditions was also monitored (green). OxDC activity under identical conditions in the absence of NO is also indicated on the MIMS plot (thin black line), showing that NO inhibition does not result merely from dioxygen depletion due to chemical re action and NO 2 formation. Reprinted from Supplementary Material of ( 54 ) with permission by the Royal Society of Chemistry. In order to test the alternate hypothesis that decreased OxDC activity resulted merely from dioxygen depletion due to the reaction of the latter gas with NO under the acidic conditions, ( 55 ) r ate of CO 2 formation at similar ly low dioxygen levels in the absence of MAHMA NONOate was measured Under these conditions the intrinsic

PAGE 65

65 OxDC decarboxylase activity was higher than that seen when MAHMA NONOate was present, ruling out this possibility ( Figure 3 9 ). OxDC inhibition by DEA NONOate A B Figure 3 10 MIMS experiments showing that OxDC inhibition is not dependent on the source of NO (A) Deoxygenated reaction buffer described in Figure 3 7 A t 2 min (during which time He was used to remove gases from the above t he reaction solution), MAHMA NONOate was added to the solution mixture (final blue ). After an additional 2 min, catalysis was initiated by the addition of 1.4 M His 6 tagged OxDC and CO 2 production was monitored ( red ). After a further 8 min, pure O 2 ( green ) was bubbled into solut ion for 30 sec onds (B) Identical experimental conditions except that NO was generated from diethyl ammonium ( Z ) 1 (N,N diethylamino)diazen 1 ium 1,2 diolate(final Reprinted from Supplementary Material of ( 54 ) with permission by the Royal Society of Chemistry C ontrol assays using an alternate NO releasing reagent DEA NONO ATE (diethylammonium (Z) 1 (N,N diethylamino) diazen 1 ium 1,2 diolate) suggested that the by product formed after NO release from MAHMA NONOate was not responsible for the observed inhibition ( Figure 3 10 ). Here, equimolar amounts of NO were generated in solution based on the respective stoichiometries d ocumented for each NONOate. The identical profile of reversible inhibition in the presence of O 2 supports the assumption that inhibition of catalysis by OxDC is due to NO.

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66 Dependence of OxDC inhibition on N ONOate c oncentration Figure 3 11 Dependence of OxDC inhibition on initial MAHMA NONOate concentration. Progress curves for the generation of 12 CO 2 under anaerobic conditions, as measured by ion current in the MIMS procedure (arbitrary units), in the pre Catalysis was initiated at t = 0 by the addition of C terminally His6 tagged OxDC. Other reaction conditions were those used to genera te the data in Figure 3 7 (Inset) An expanded plot of the data generated during the first minute of reaction using a linear axis to emphasize differences in magnitude of 12 CO 2 production. Reprinted from ( 54 ) with permission by the Royal Society of Chemistry. Varying the initial amount of MAHMA NONOate under identical experimental conditions est ablished that the extent of OxDC inhibition was dependent on NO concentration, consistent with an NO binding site on the enzyme ( Figure 3 11 ). Initial plateau in the progress curves is the response time of the instrument as described in Chapter 2. Mode of Inhibition of NO A measure of initial velocities in reactions with varying concentrations of substrate and NO showed a distinct uncompetitive mode of inhib ition by the convergence of line s on the ordinate (K m /V max ) of Figure 3 12 The data at the largest c oncentration of NO did

PAGE 67

67 not adhere well to the magnitude of values projected by the global fit in Figur e 3 12 This was possibly due to the experimental limits of detecting lower initial rates, which may already be taking place within the response time of t he instrument Alternatively, higher concentrations of NO may result in hitherto undetected [ESI] forms of the enzyme, manifesting in a deviation of the fit to the standard uncompetitive inhibition equation. Regardless, the v 4 fitted data we re consistent w ith an uncompetitive mode of inhibition with a derived K i of 40 1 M, which is of a similar magnitude to the K m for O 2 of 28 8 mM (measured from the dependence of decarboxylase activity on dioxygen concentration) ( 14 ) Th is indicates that NO inhibits OxDC by targeting a form of the enzyme when substrate is present. It may further suggest that dioxygen either binds after binding of substrate, or that the putative O 2 pocket is more accessible after substrate binds to the enzyme. Figure 3 12 Mode of Inhibition of NO. Hanes Woolf plot of single measurements in OxDC catalytic rates measured from changes in m/z 45 ion currents ( 13 CO 2 ) in oxygen depleted reactions containing 0.2% Triton X, 1 10 mM o f 13 C 2 oxalate, 1.44M C terminally His 6 tagged OxDC in the presence of (black) 0, (blue) 10, (green) 25 and (red) 66 M DEA NONOate,buffered at pH 4.3 with 50 mM sodium acetate. Solid lines are v 4 weighted global fits of all data, which fit best to an Unc ompetitive mode of inhibition model.

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68 In hibition of NO on catalysis by s ite specific mutants of OxDC Figure 3 13. Effect of NO on catalysis by untagged C 383S OxDC mutant. Time course of changes in ion currents (arbitrary units), determined using MIMS for 1 2 CO 2 ( red ), NO ( blue ) and O 2 ( green ). Full experimental details are described in F igure 3 7 except that 0.43 M C383S OxDC was used to initiate catalysis and pure O 2 was bubbled in for 30 seconds at t = 12 minutes Reprinted from Supplementary Material of ( 54 ) with permission. Given that NO dependent OxDC inhibition could be reversed by the re introduction of dioxygen, it was postulated that the NO inhibited decarboxylase activity by binding to the Mn(II) center, possibly at a coordination site occupied by dioxygen during catalytic turnover, ( 22 ) or by nitrosylating the side chain of Cys 383 in the protein, ( 56 ) which is locat ed at the C terminus distant from the putative catalytic site ( 9, 10 ) The latter proposal was ruled out, however, by the fact that the C383S OxDC mutant exhibited identical catalytic behavior to the wild type enzym e, including reversible inhibition by NO under the conditions of our earlier experiments ( F igure 3 13 ) C383 is the only cysteine in the amino acid sequence of B.subtilis OxDC and is three residues away from the C terminal end ( Figure B 3A) Furthermore, t he possibility that observed inhibition by NO is due to interactions with the C terminal polyhystidine tag was likewise eliminated by the fact that the C383S OxDC mutant had no polyhistidine tag This was confirmed by

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69 identical results between native wild type OxDC and C terminally His 6 ta gged OxDC in the presence of NO. Inhibition caused by covalent modification of tyrosine residues as observed in Mn dependent superoxide dismutase, seemed unlikely since such modifications would be irreversible, even in th e presence of abundant O 2 Regardless, similar experiments w ere conducted on the Y200F OxDC mutant whose mutation replaces the only conserved tyrosine found to affect decarboxylase activity ( Sh ukla, M. unpublished ) Y200F only possessed 1/ 4 th the activity of wild type. Contrary to the hypothesis that this mutant would be unaffected by the presence of NO, reversible inhibition in the presence of O 2 was observed to be identical to that of wild type (Figure 3 14) Figure 3 14 Effect of NO on catalysis by Y 200F OxDC mutant. Time course of changes in ion currents (arbitrary units), determined using MIMS for 1 2 CO 2 ( red ), NO ( blue ) and O 2 ( green ). Full experimental details are described in F igure 3 7 except that catalysis was initiated by 1.0 M Y200F ( Shukla M. unpublished ) OxDC mutant

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70 Figure 3 15 Effect of hemoglobin on catalytic inhibition by NO. Time course of changes in ion currents (arbitrary units), determined using MIMS for 1 2 CO 2 ( red ), NO ( blue ) and O 2 ( green ). Full experimental details are describ ed in F igure 3 7 except that NO was generated from 35 M DEA NONOate, 50 Ls of hemoglobin was added at 12 minutes instead of air, and catalysis was initiated by 0.3 M C terminally His 6 tagged OxDC. O 2 and CO 2 from the hemoglobin aliquot is indicated by th e rise in these signals at t = 12 minutes. Detected catalytic CO 2 (red) recovered is concomitant to the plateau of exogenous O 2 (green). Observed lag time preceding the reversal of inhibition was a consistent feature in these NO experiments and was further observed to be indirectly dependent on the amount of O 2 introduced into the inhibited system (ie. the more O 2 introduced, the shorter the time lag prior to the recovery of catalytic CO 2 ) F urthermore, the alternative addition of hemoglobin over air/oxygen to the NO inhibited system similarly resulted in the revers al of inhibition but without the time lag (Figure 3 1 5 ). The ability of hemoglobin to more rapidly recover catalytic CO 2 confirm s a reversible non covalent interaction of molecular NO within the enzyme, ruling out a suspected inhibitory NO adduct which would otherwise be unaffected by the presence of hemoglobin. This further suggest s that the observed time lag preceding the revers al of NO inhibition by O 2 is more likely

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71 due to a c hemical reaction with enzyme bound NO rather than simple displacement of it In order to more thoroughly verify the effect of hemoglobin on enzyme bound NO, a similar exp eriment was repeated (Figure 3 16 ) where the NO inhibited system was purged of remaining NO in solution by the bubbling in of nitrogen gas (N 2 ). Simila r to an earlier experiment (Figure 3 8) where OxDC remained inhibited after the removal of NO from solution, it was only in the addition of hemoglobin (at t = 15 minutes) where catalytic CO 2 was regenerated, as indicated by a note worthy rise in the red signal starting from ~ 3 x 10 9 A. Initial spike in CO 2 upon injection of hemoglobin is due to the introduction of CO 2 from the hemoglobin aliquot. Nonetheless, catalytic recovery in this experiment was n ot prec eded by the usual lag time observed when the inhibition was reversed by oxygen. Figure 3 16 Effect of hemoglobin on catalytic inhibition by NO after bubbling with N 2 Same experiment as in Figure 3 1 5 except that N 2 gas (cyan) was bubbled into the inhi bited system at t = 10 minutes. Recovery of catalytic CO 2 was observed only after addition of hemoglobin at t = 15 minutes.

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72 Constant Wave Electron Paramagnetic Resonance (CW EPR) Experiments Figure 3 17. Overlaid CW EPR spectra of the Mn (II) centers in OxDC in the presence and absence of NO released from MAHMA NONOate.(A) Degassed (Ar) sample of WT OxDC in 110 mM acetate buffer, pH 4.1 (black). (B) Same sample as in (A) to which MAHMA NONOate was added (red). (C) Degassed (Ar) sample of WT OxDC in 110 mM acetate buffer, pH 4.1, containing MAHMA NONOate and oxalate (green). (D) Metal free 110 mM acetate buffer, pH 4.1, containing released NO (blue), and the difference spectrum (purple) generated by subtraction of (D) from (B). All spectra were recorded at 4.2 K Reprinted from ( 54 ) with permission by the Royal Society of Chemistry. A series of EPR measurements were performed on frozen solutions of OxDC containing NO with the expectation that NO might bind to one, or both, of the high spin Mn(II) centers present in OxDC ( 17 ) As a result, it was anticipated that th e intensity observed for the EPR signal(s) associated with the Mn(II) centers would decrease because the interaction of NO with the enzyme would yield a Mn(III) species possessing wild type OxDC (0.12 mM) was dissolved in 110 mM KOAc buffer, pH 4.1, which had been carefully degassed by sparging with argon (120 mL total volume). After freezing in liquid isopentane pre cooled in liquid N 2 CW EPR spectra were taken at 4.2 K ( Figure 3 1 7 ). The initial degassed OxDC sample showed the usual X band spectrum for the bound Mn(II) ions, ( 10 ) i.e. six relatively sharp lines exhibiting the average hyperfine coupling

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73 constant of 92 G typica l of hexa coor dinated manganese ( 57 ) In addition, the spectrum shows a shoulder at 3050 G and a broad maximum centered at 2340 G. Using multi frequency EPR measurements, we have interpreted this spectrum as arising from two different Mn(II) sites with fi ne structure parameters of |D|=1200 MHz an d 2150 MHz ( 17 ) Recent work has suggested that the X band EPR spectrum of the enzyme is likely sensitive only to the Mn(II) site possessing the sma llest fine structure constant D. This sample was then mixed with 0.5 mM MAHMA NONOate ( full details are in the E xperimental section ) and we examined whether there was a change in the intensity of all, or parts of, the Mn(II) signal when OxDC was exposed to NO. Perhaps unexpectedly, the Mn (II) signal in the EPR spectrum of OxDC under these conditions was of approximately the same intensity as that observed in the absence of NO ( Fig ure 3 1 7 ). Although there was some background signal from MAHMA NONOate (presumably arising from free NO), subt raction of the relevant spectra showed a reduction in Mn(II) signal intensity of only 10 20% in conditions under which NO completely inhibited the decarboxylase activity ( Figure 3 1 7 ). Such a decrease is expected to be associated solely with OxDC sample di lution resulting from addition of the MAHMA NONOate solution. A sample of OxDC that contained both MAHMA NONOate and oxalate also gave similar results when analyzed using our standard EPR procedures ( Figure 3 1 7 ). These observations seem to indicate that N O does not exert its inhibitory effect on decarboxylase activity by displacing dioxygen from the metal center or by forming a heptacoordinate Mn(II) species, at least for the site that is visible in the X band EPR spectrum. High frequency EPR analysis of O xDC by other workers

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74 has also suggested that the Mn(II) site exhibiting the smaller fine structure value D is actually located in the N terminal domain of the enzyme ( 16 ) this assignment being based on spectroscopic evidence for the presence of a pentacoordinate Mn(II) center in OxDC at high solution pH ( 10 ) If this interpretation is correct then our findings would seem to exclude NO binding at the solvent accessible catalytically active N terminal site ( 9 ) These observations are also consistent with the remarkably small number of well characterized mononuclear {Mn NO} complexes that have been reported ( 58 60 ) We also note that only circumstantial ev idence for the formation of Mn(III) during catalytic turnover (a metal species that might bind NO) has been reported ( 61 ) On the other hand, it remains possible that any Mn(II)/NO interaction is masked by the compl exity of the X band EPR spectrum for OxDC. ( 16, 17 ) Experimental Section Materials (Z) 1 (N methyl N [6 (N methylammoniohexyl)amino]diazen 1 ium 1,2 diolate (MAHMA NONOate) and diethylammonium (Z) 1 (N,N diethylami no)diazen 1 ium 1,2 diolate (DEA NONOate) were obtained from Cayman Chemical (Ann Arbor, MI). and 13 C 2 oxalic acid (99%) was purchased from Cambridge Isotope Laboratories (Andover, MA) DNA primers bearing the C383S point mutation, NdE, and BamHI restricti on sites were designed and purchased from Integrated DNA Technologies, Inc. (Coraville, IA). DNA sequencing services were done by the core facility of the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. His6 tagged Oxalate Decarboxylase Expression and Purification Protein samples were prepared a s d escribed in C hapter 2

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75 Untagged Recombinant W ild type and C383S OxDC Mutant The gene encoding the C38 3S OxDC mutant was obtained by PCR using the wild type B subtilis Yvrk /pET9a construct as template. A forward primer containing the the NdE1 restriction s equence ( GGA GGA AA C ATC ATA TG A AAA AAC AAA ATG was used in tandem with a revers GCA TCA GGA TCC TTA TTT ACT GC T TTT CTT TTT CAC TAC teine to serine mutation and the BamHI restriction sequence The resulting amplicon was digested with the NdE1 and BamHI restriction enzymes, giving a DNA fragment similarly digested kanamycin resistant pET9a vector into which it was ligated using T4 ligase. The resulting C383S plasmid construct was cloned and purified from JM109 E coli using the Promega plasmid purification kit and was submitted for DNA sequencing Upon confirmation of the desired gene sequence the C38 3S OxDC construct was used to transform BL21/DE3 E.coli for protein expression. Both of the untagged enzymes were then expressed and purified using a slight modification of published procedures (Appendix A) ( 18 ) in which fractions from hydrophobic interaction chromatography on phenyl sepharose were exhaustively dialyzed against a storage buffer of 50 mM Tris buffer, pH 8.5, or 20 mM Hexamethylenetetramine pH 6.0, containing 500 mM NaCl Membrane Inlet Mass Spectrometry (MIMS) Description of the instrument and s ettings are as described in C hapter 2 Reaction Mixtures Unless otherwise stated, reaction solutions initially contained either unlabeled 50 mM potassium oxalate or 50mM 13 C 2 labeled oxalate (99%) diss olved in in 50 mM acetate buffer, pH 4.2, containing 0.2% Triton X 100 and a trace amount of Antifoam A (final volume 2 mL). Because generated NO rapidly gets oxidized in solution, d ioxygen

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76 was then depleted in these mixtures by purging with He until MIMS analysis showed baseline levels. In the absence of NO, reactions were initiated by the addition of wild type, His 6 tagged OxDC or the C383S OxDC mutant so that the final enzyme In experiments examining the ability of NO to inhibit OxDC activity, a solution of M NO) ( 4 6 ) or 10 mM diethylammonium ( Z ) 1 (N,N diethylamino) diazen 1 ium 1,2 diolate (final concentration ( 47, 48 ) dissolved in 0.01 M aq ueous NaOH was injected into the reaction vessel 2 min prior to initiation of the reaction by the addition of enz yme. Reversibility of NO dependent OxDC inhibition was investigated, by later addition 50 Ls hemoglobin extracted from human blood plasma, or bubbling of either CO 2 scrubbed air (15 30 sec) or pure O 2 (30 sec) into the NO inhibited enzyme reaction soluti on Continuous Wave EPR Measurements An Tris buffer, pH 8.5, containing 500 mM NaCl) was mixed with 2 M potassium acetate small a mount of enzyme that precipitated on lowering the solution pH was then removed by centrifugation (14,850 rpm, 5 min) and the resulting clear supernatant was collected (pH 4.5) and employed in subsequent EPR experiments after degassing with argon (care was taken to prevent excessive foaming) for 1 min in an anaerobic chamber (dry box flooded with N 2 gas) under a nitrogen atmosphere. This procedure reduced the solution volume to 240

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77 transferred to a quartz tube (3 mm internal diameter and 4 mm external diameter), which was capped and rapidly frozen in cold isopentane (pre cooled to near its freezing point in liquid N 2 ). The cold sample tube was placed in a pre cooled cryostat (Oxford ESR900) for CW EPR measurements (Figure 3 11 spectrum (A)). After subsequent thawing of the degassed sample (in the anaerobic chamber), the tube was opened under N 2 (g) atmosphere prior to ad dition of the NO donor. 13 MAHMA NONOate in 10 mM aq. NaOH was then added to the enzyme containing solution in the EPR tube and allowed to react for 5 min before the sample tube was re capped and rapidly frozen using pre cooled isopentane. CW EPR measurements we re then performed as before (Figure 3 1 7 spectrum (B)). After thawing, the solution pH of oxalate solution in acetate buffer, pH 4.1) was added as well as 1 mM MAHMA NONOate prior to cooling and CW EPR measurements (Figure 3 1 7 spectrum (C)). A so prepared using 20 mM metal free Tris buffer, pH 8.5, which was subjected to identical treatment as the NO exposed solution of OxDC (Figure 3 1 7 spectrum (D)). Additional control CW EPR spectra were obtained for the 20 mM metal free Tris buffer, pH 4.1, and a solution of MAHMA NONOate in 10 mM aq. NaOH. These experiments were designed to reveal the potential binding of dissolved NO to those Mn sites which were accessible to X band EPR analysis through changes in their fine structure values.

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78 All EPR spec tra were recorded at liquid helium temperature, using a commercial BrukerElexsys E580 spectrometer with the standard rectangular TE102 resonator. Instrumental parameters were: 90 kHz modulation frequency, 15 G modulation amplitude, 0.6 mW microwave power, 9.4347 GHz microwave frequency, 330 ms time constant, and 330 ms conversion time/point. Each spectrum consisted of a single scan of 5001 data points over a scan range of 50 to 7050 G

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79 CHAPTER 4 SMALL A N ION INHIBITION OF CATALYSIS BY OXDC Introduction Dis covery and study of enzyme inhibitors are important in elucidating key structures in the catalytic mechanism For the purpose of exploring medical applications of OxDC identification of inhibitors become critical in assess ing enzymatic behavior outside of its native source or environment. Reported inhibition of nitric oxide as measured by MIMS had opened a new realm of possibilities in the application of MIMS on the study of OxDC. To date nitric oxide is the only reported inhi bitor for Bs OxDC. Although ear lier studies on the fungal OxDC have mentioned effects of compounds such as sodium azide, sulfite and dithionite ( 37 ) on catalytic activity, further characteriz ations on these compounds were limited by the methodologies then Becau se of the direct and real time nature of the MIMS assay, i t was now important to employ the method to further investigate the OxDC reaction, in terms of possible product inhibition, and inhibitory effects of molecular analogs of substrate, product and co f actor O 2 Through this method and its ability to utilize iso tope labeled substrate to distinguish catalytic from non catalytic carbon diox ide in solution, an array of small molecule inhibitors have been examined for Bs OxDC to an extent that was difficult t o achieve with previously publi shed m ethods. Results and Discussion An example of the data provided by MIMS is given in Figure 4 1 for a series of CO 2 progress curves showing inhibition of OxDC by sodium azide at pH 4.2. Initial velocities were determined from the slopes at 5% to 10% of completion and plotted in F igure 4 1 for later discussion under the section on azide.

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80 Figure 4 1. Inhibition by azide ions of catalysis by OxDC. Progress curves of accumulated CO 2 in solution from the catalysis of 10 mM o xala OxDC in the presence of (black) 0 mM, (blue) 2.5 mM, (orange) 5 mM, (green) 15 mM, and (red) 40 mM sodium azide in a reaction buffer containing 0.2% Triton X 100 and 100 mM sodium citrate at pH 4.2 and maintained at 25 o C Catalytic P roducts In order to assess the inhibition of products carbon dioxide and formate on catalysis by OxDC, catalytic rates were monitored in reaction buffers pre equilibrated with 25 2 00 mM sodium formate; or saturating levels of dissolved CO 2 bubbled into solutions c ontaining 13 C 2 labeled oxalate substrate. Experiments showed that CO 2 itself did not inhibit OxDC as indicated by unaffected rates of catalytic 13 CO 2 formed in solution s containing saturating levels of dissolved CO 2 Rates of CO 2 formation in the presence of 200 mM formate were reduced by 40% comp ared with the absence of it indicating that formate is not a significant inhibitor (K i > 200 mM). These results show that catalysis is not significantly blocked by the buildup of either product in solution. Utiliz ing a simil ar approach, an array of different anions were surveyed for effects on catalysis by monitoring the initial rates of catalytic CO 2 formation by wild type OxDC

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81 in the presence of 10 mM oxalate (near K m ) and 0 200 mM anion. Any anion suppressing the rate of CO 2 formation by at least 50% were further s urveyed by gathering initial rates at additional intermediate concentrations within the broad 0 100 mM range. A rough preliminary estimate for each K i was de rived by plotting initial rates of CO 2 form ation vs. concentration of eac h anion Data points were fitt ed to the equation v = V max / (1 + ([I] / [K i ] ) ). is max anion. Figure 4 2. Effects of dissolved halides on catalysis by OxDC Single measurements of i nitial velocities of generated CO 2 from OxDC catalysis conducted in the presence of 0 mM, 100 mM, 200 mM ( blue ) NaCl, ( red ) NaBr; or 2.5 mM, 10 mM, 3 0 mM, 100 mM, and 200 mM ( black ) KI dissolved in solution. Reaction mixture also contained 0.2% Triton X 100, 10 mM potassium oxalate, and 100 mM sodium citrate buffered at pH 4.2 and maintained at 25 o C. Black curve ) represents data fitted to f(x) = V max / [1 + ( [ I ] / K i ) ] with an R 2 value of 0.985. Derived estimated K i for iodide was 51 7 mM Error bars indicate the 3 % error in deriving the slope s from the linear fitting of the initial 5 10% of the re spective progress curves of product formation. A good inhibitor would ideally be one with an estimated K i which is lower than the K m of OxDC (4 mM) However, b ecause no inhibitor has ever been characterized for this enzyme prior to nitric oxide, any anion resulting in an estimated K i of less than 5 0 mM

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82 were of interest. These anions were further characterized for their mode of inhibition as previously done with NO by varying inhibitor concentration around the estimated K i and substrate concentrations around the previously observed Km for oxalate which was at 4 mM Anionic Buffer E ffects In order to ensure that any buffer used in the reaction mix did not contribute to any effect observed on catalytic generation of CO 2 each buffer was first surveyed by runnin g control experiments with wild type enzyme an d 10 mM oxalate in varying buffer concentrations (50 200 mM) Among these buffers (acetate, citrate, piperazine) acetate was the only one to show any inhibitory effect at pH 4.2; however, very weak with an es timated K i >100 mM. Piperazine buffer which was used for reactions at pH 5.4 and pH 6.0 showed no effect on catalytic activity up to 200 mM. Small anionic halides were also surveyed. Catalysis was not significantly affected by bromide ions up to 100 mM co ncentrations. Iodide demonstrated some inhibition with K i near 50 mM (Figure 4 2). In the same figure, chloride ions which are inherent to the storage buffer of the enzyme showed no effect on catalytic activity. Counterions of the anionic salts used in thi s chapter had no effect on catalysis by OxDC. This was confirmed by unaffected catalytic rates in separate wild type experiments performed in reaction buffers containing 200 mM sodium chloride, 200 mM potassium chloride, and 200 mM ammonium chloride. Diffe rent chloride anionic salts were used upon showing in earlier experiments that chloride had no effect on catalysis. Phosphate measured at pH 6.0 had a small inhibitory effect on catalysis with K i near 50 mM. By contrast, sulfate anions measured at pH 4.2 h ad no effect at all up to a concentration of 200 mM

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83 Nitrate and Nitrite Figure 4 3. Inhibition by nitrite ion (NO 2 ) of catalysis by OxDC. Hanes plot of initial catalytic rat es usi mM, (green) 2 m M, and (red ) 4 mM sodium nitrite at pH 4.2. Lines represent data fitting t o an uncompetitive model (R 2 = 0.967) using v 4 algorithm with K i for nitrite estimated a t 1.3 0. 2 mM. Because of the reported dependence of catalysis on the presence of oxygen, eff ect of oxygen analog nitric oxide on OxDC catalysis was explored in C hapter 3 T he reported reversal of NO inhibition by abundant oxygen was presumed to be due to a reaction between supplied O 2 and enzyme bound NO. This idea was consistent with the reporte d oxidation of nitric oxide in acidic solution ( 49 ) Oxidation products nitrate (N O 3 ) and nitrite (NO 2 ) anions in solution, had different effects on catalysis. Nitrate (from either potassium or ammonium salts) showed no effect on the rate of CO 2 generated by OxDC catalysis up to a concentration of 200 mM. Nitrite by contrast, was inhi bitory, with a similar uncompetitive mode of inhibition as NO, but at a weaker magnitude (K i ~ 1.3 mM). Nitrite forms very small amounts of NO at acidic pH (estimated to form NO at rates less than 1 nM/sec under the conditions of Figure 4 3 ( 62 ) However, levels of NO in

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84 solution, measured by its m/z 30 peak, showed that NO was not involved in this inhibition. Again, the uncompetitive mode suggests an interaction of nitrite with a species of the enzyme formed after the binding of substr ate. From the proposed catalytic scheme in Chapter 1, the nonlinear nitrite anion may be an analog of the formate radical anion intermediate, binding at the formate site in the described structure ( Figure 1 5 ) Inhibition by nitrite prompted us to look int o the surveyed anions not only f rom the perspective of size, but more importantly, from the perspective of geometry. Wh ich anions were more structurally analogous to substrate, product, or cofactor? And what effects did they have on c atalysis? These questi ons came from the fact that linear NO was significantly inhibitory though it was an uncharged molecule. Nitrite, however similarly anionic as oxalate ( substrate ) and formate ( product ) possessed a bent geometry. Bicarbonate We studied bicarbonate to estima te inhibition in a separate set of experiments buffered at pH 6.0 to enhance bicarbonate concentration. Experiments showed bicarbonate to be the only competitive inhibitor among the small molecules investigated here, as determined by the parallel lines of F igure 4 4 The entire data set was fit using a competitive model and v 4 weights, and again there was deviation from the competitive model for the largest concentration of bicarbonate ( Figure 4 4 ). The equilibrium between dissolved carbon dioxide and bicar bonate (pK a is 6.3 5 for the equilibrium [H + ][HCO 3 2 ]); hence, only a fraction ( 0. 46 ) all species of CO 2 exists as bicarbonate at the pH 6.0 of this experiment. Taking this into consideration, the K i for bicarbonate derived from the data of Figure 4 4 is 1.1 0.1 mM

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85 Bicarbonate at chemical equilibrium exists at about 1% of the concentration of CO 2 in solution at the pH 4.2 used in the experiments in ( Figures 4 1, 4 3, and 4 5 ) As shown in Figure 4 1, we measured concentrations of CO 2 less than 5 mM in these experiments. Bicarbonate formed from this product CO 2 would accumulate to a maximum of 0.05 mM. With the K i for bicarbonate near 1 mM cited above, we estimate that there is negligible inhibition by bicarbonate affecting our results in Figures 4 1 4 3, and 4 5 Figure 4 4. Inhibition by bicarbonate of catalysis by OxDC. Hanes plot of initial catalytic (orange) 4 mM, (green) 6 mM, and (red) 8mM potassium bicarbonate. Other comp onents were 0.2 Triton X, and 100 mM piperazine at pH 6.0. Solid lines represent the fit of all data to a competitive inhibition model using a v 4 algorithm with K i for bicarbonate at 1.1 0.1 mM. R 2 = 0.999 Bicarbonate caused a competitive inhibition of c atalysis with K i near 1 mM ( Figure 4 4 ). Competitive inhibitors are often associated with substrate analogues, and bicarbonate is only very loosely analogous with the structure of oxalate. Formate and acetate (Ki >100 mM) are better analogs but are very we akly inhibitory. Glycolate, an

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86 even closer analog of oxalate than formate and acetate, is not inhibitory to the enzyme (unpublished data). These features are consistent with the known high specificity of OxDC in catalysis. The competitive inhibition by bic arbonate then emphasizes a possible effect of the third oxygen atom of bicarbonate, which is lacking in formate, acetate, and glycolate. This third oxygen atom may overlap an oxygen atom of the cofactor O 2 in the uninhibited pathway, structure B of Scheme 1 ( 23 ) These data raise an interesting possibility that bicarbonate may be a product of the catalysis by OxDC. Th e half life for the uncatalyzed conversion of bicarbonate to CO 2 at pH 4 is near 0.1 s so substrate bicarbonate would not be detected in our experiments. None of these mechanisms currently proposed for OxDC suggests bicarbonate as a product ( 5 ) Azide and Thiocyanate Unlike the observed effects from halides, which were initially perceived to be small enough to access the catalytic site, considerable inhibition was observed from linear anions such as thi ocyanate and a zide.(Figure 4 1) From these anions, we have observed yet a third mode of inhibition. The data for azide inhibition are best fit to a noncompetitive model as demonstrated by the convergence of lines on the abscissa of the Hanes plot of Figure 4 5 These l ines represent a fit of the entire data set of Figure 4 4 to the noncompetitive model using v 4 weighting, as described in the Experimental section ( 50, 52 ) It was a feature of several of our inhibitor s that the highest inhibitor concentration used did not adhere well to our models. This feature is exaggerated by the emphasis of v 4 weighting on the points representing higher initial velocities and the nature of the Hanes plot containing reciprocal veloc ities on the ordinate. Nevertheless, this poor fit suggests more complex modes at higher concentrations of these anions, perhaps due to the effect of secondary binding sites. A fit of the data of Figure 4 5 to a

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87 noncompetitive model using v 4 weighting gave K i = 14 9 mM when we neglected the data at the highest inhibitor concentration. Inhibition by thiocyanate was also determined to be best described as noncompetitive with K i determined near 4 mM. Figure 4 5 Mode of i nhibition by azide of catalysis by OxDC. Hanes plot of initial black ( blue ) 2.5 mM, ( orange ) 5 mM ( green ) 15 mM, and ( red ) 40 mM sodium azide at pH 4.2. Solutions were as described in Experimental section Data points are the le ast squares slopes of the initial velocity of single experiments as shown in Figure 4 1 Lines represent a fit of a ll the data to a noncompetitive inhibition model using a v 4 algorithm with K i for azide at 14 9 mM There are at least two possibilities in explaining the noncompetitive mode: 1) that these anions interact with the enzyme at a location other than that for substrate; 2) or that these anions bind to and inhibit a species of the enzyme that is different from that to which substrate binds. As an example of the latter, the dissociation of product formate may leave the enzyme in an inactive form requiring conformational change or reduction of Mn(III) to become active. The binding of azide to the inactive form would result in a noncompetitive inhibit ion.

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88 It is interesting that B. subtilis OxDC has coordinating amino acid ligands of the catalytic manganese similar to that of manganese superoxide dismutase (MnSOD) (three histidine residues and a carboxylate of Glu for OxDC ( 9 ) and Asp for MnSOD ( 63 ) However, MnSOD i s characterized predominantly as Mn(III)SOD which as a d 4 ion prefers a pentacoordination; in fact, MnSOD has a single solvent molecule in the inner sphere of coordination ( 63 ) In contrast, OxDC is isolated predominantly as Mn(II)OxDC which as a d 5 ion prefers to coordinate a sixth ligand and is characterized as a hexacoordinate with two solvent molecules in the inner sphe re ( 9, 10 ) O f the many anions and NO investigated here, only azide has been reported to inhibit MnSOD binding to Mn(III)SOD in a predominantly outer sphe re complex at room temperature ( 64, 65 ) In contrast substrate analogs such as azide bind directly to the metal in Mn(II)SOD, consistent with the property of the d 5 configuration to bind lig ands more tightly to the metal ( 66, 67 ) By analogy we may expect azide to bind directly to the manganese of OxDC. The active site cavity of MnSOD is more steric ally constrained than for OxDC ( 9, 63 ) which may explain why OxDC is susceptible to inhibition b y a wider range of anions. An oxidized metal of Mn(III)OxDC has not been observed in the wild type Future EPR experiments have been proposed to investigate whether or not azide binds to the Mn(II) of OxDC. If the inhibitory complex involves azide bound di rectly to the metal of Mn(II)OxDC displacing the solvent ligand, the resulting noncompetitive inhibition implies that the substrate does not bind directly to the metal. The binding of azide to E. coli MnSOD has a binding constant near 7 mM ( 67 ) near the value of 14 mM found for OxD C (Figure 4 5). The binding of azide to oxalate

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89 oxidase, which has an active site very similar to OxDC ( 8 ) is also in the millimolar range ( 68 ) Experimental Section His6 tagged Oxalate Decarboxylase Expression and Purification Protein samples were prepared as described in C hapter 2 Membrane Inlet Mass Spectrometry (MIMS ) Description of the instrument an d settings are as described in C hapter 2 Reaction Mixtures Unless otherwise stated, all 2 mL reaction mixtures contained 0.2% Triton X 100, 1 10 mM ( 13 C 2 labeled or unlabeled) potassium oxalate, and 50 100 mM of buffer at pH 4.2 6.0 Buffers used were 50 mM acetate or 100 mM citrate for experiments performed at pH 4.2; and 100 mM p iperazine for those performed at pH 6.0 Prior to catalysis, reaction mixtures were either equilibrated by the bubbling of CO 2 free air, or deoxygenated by the bubbling of helium gas. However, catalyses were initiated by the addition of (5 20 Ls) air equilibrated enzyme stocks in order to provid e the oxygen that is reported necessary for catalysis ( 7 ) Reactions were initiated by the addition of 0.3 1.44 M OxDC and maintained at 25 o C throughout each expe riment by a circulating water bath. Stock solutions of double labeled carbon 13 oxalate ( 13 C 2 labeled oxalic acid; 99% 13 C; Cambridge Isotope Laboratories Andover, MA) were prepared by dissolving the pre weighed solid and adjusting the pH to 4.2 with min imum amounts of KOH. Anion Inhibition Unless otherwise noted, the following were tested at pH 4.2 as possible anionic inhibitors from 0.5 1.0M stocks : 50 200 mM sodium pyruvate, 50 200 mM sodium

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90 formate, 100 200 mM potassium nitrate, 50 mM ammonium nitrate, 100 200 mM sodium sulfate, 5 40 mM sodium azide, 50 mM sodium cyanate, 0.75 50 mM sodium thiocyanate, 1 50 mM sodium bicarbonate (at pH 6.0), 100 200 mM sodium chloride, 100 200 mM sodium bromide, 2.5 200 mM potassium iodide, 20 2 00 mM potassium phosphate (at pH 6.0), 50 200 mM sodium citrate, and 50 200 mM sodium acetate. Solutions containing these ions, except bicarbonate, were deoxygenated by bubbling of helium prior to each experiment. Experiments using bicarbonate were car ried out by the addition of sodium bicarbonate to degassed buffer solutions one minute prior to the initiation of catalysis. Inhibitory effects were detected as reduced rates product formation measured as ion currents for m/z 44 ( 12 CO 2 ) or m/z 45 ( 13 CO 2 ) i n catalytic mixtures containing varying concentrations substrate and inhibitor. Initial Rates Calculations for initial rates of catalysis and associated errors are a s described in Chapter 2. Mode of Inhibition and Statistical Estimation of Parameters The m ode of inhibition and statistical estimation of inhibition constants were determined by a least squares, best fit approximation using v 4 weighting factors ( 50, 52 ) where v is initial velocity. Data we re globally fitted to a kinetic model for simple competitive, simple noncompetitive, and simple uncompetitive inhibition ( 69 ) to derive a best fit estimate of the inhibition cons tant. In each of the Hanes plot s showing inhibition, e ach data point represents a single measurement of initial velocity. The solid lines on the Hanes plots represent the calculated values of v using the parameters estimated by the least squares procedures.

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91 CHAPTER 5 CO N CLUSIONS AND FUTURE WORK MIMS and OxDC Catalysis MIMS provides a direct continuous, sensitive, and real time assay to monitor decarboxylase activity, yielding results comparable to those acquired from tradi tional indirect endpoint assays I mportantly, MIMS provides the added feature of simult aneously monitoring other gaseous species providing, for example, a concomitant measure of oxidase activity of OxDC. MIMS measures isotopically labeled products providing a wide flexibility in monitoring effects on reaction catalysis such as addition of in hibitors and changes in reaction conditions This renders MIMS a versatile tool in s tudying enzymic catalysis in CO 2 generating reactions Study of the r eversible inhibition of NO as an analogue of dioxygen was a considerable step forward in further underst anding OxDC catalysis and assessing the value of the MIMS assay. The ability to narrow down the inhibitory form of NO on OxDC from a range of possibilities was not trivial, considering its limited stability in acidic solution and its potential reactivity w ith known radical intermediates formed in OxDC catalysis. The Interesting uncompetitive mode of inhibition determine d through this approach provides useful suggestions on the possible order of binding of substrate and oxygen cofactor if not conditions in which the putative oxygen pocket is more accessible Furthermore, it provide s more information for optimizations on crystallographic approaches in determining the yet unelucidated Michaelis Menten complex of OxDC An actual look into the suspected product inhibition of OxDC from the perspective of formate and carbon dioxide was a n oteworthy achievement delivered by MIMS. The

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92 ability to utilize this method to screen for inhibitors based on structural and size homol ogy with substrate, product and cofactor sup ports the speculated high specificity of the enzyme. The different modes of inhibition observed here for catalysis by OxDC emphasize the complexity of this catalysis and suggest further experiments to identify inhibition targets and multiple forms of the e nzyme required to carry out catalysis. The exact locations of inhibitor binding and enzyme species to which they bind are uncertain at this point. However, the implications of these inhibition studies provide vital ideas in optimizing further crystallograp hic and spectroscopic experiments investigating the key steps in the mechanism of OxDC. Insights on Future Work Findings reported in this work have demonstrat ed the potential of the MIMS assay on the continued study of OxDC. O ptimized experimen tal conditio ns in this work have facilitated preliminary work on other aspects of the enzyme; thus laying the groundwork for continued endeavors in better understanding OxDC and its catalytic mechanism. Michaelis Menten Complex of OxDC Implications of C hapter 3 on nit ric oxide provide a wellspring of information for studying the Michaelis Menten complex of OxDC. Although it is known that oxalate binds to the catalytic site and that dioxygen is required, very little is known on the mode in which substrate binds. Further more, it also remains a question as to where the dioxygen cofactor binds and the role it plays in catalysis. Current ideas on the mechanism pictures the M ichaelis M enten complex as limited to the N terminal Mn(II) site. If NO do es not directly interact wit h the N terminal Mn(II), which is responsible for the unperturbed smaller fine structure in the cw EPR spectra of NO inhibited OxDC, then a picture of the Michaelis menten complex may either include the C terminal Mn(II)

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93 or a region much wider than the con straints of the N terminal active site. Since the uncompetitive inhibitors with the lowest K i s were NO (40 M) and its oxidation product anion NO 2 (2 mM), attempts in crystallizing the MM complex may be facilitated by experimental conditions parallel to t he nonaerobic buffer conditions of the MIMS experiments in the presence of oxalate substrate. Competitive inhibition by bicarbonate (K i ~ 1 mM) albeit at a slightly elevated pH, likewise opens doors for potentially utilizing 13 C labeled bicarbonate and EPR spectroscopy in determining the binding mode of the inhibitor. Although bicarbonate more closely resembles formate product than oxalate substrate, the fact that it targets the same form of the enzyme as oxalate presumably during turnover may give structur al insights on the early stages of catalysis. Oxygen binding site and Oxygen Dependence Earlier described experiments on the reversal of nitric oxide inhibition on OxDC by the addition of hemoglobin were vital in showing that molecular NO inhibited the enz yme and not a reaction product/adduct with the enzyme. However, further refinements and additional experiments are necessary to more clearly demonstrate that reversal of inhibition was a result of enzyme hemoglobin in solutio n, which is presumed to have a higher affinity for it. Since hemoglobin rapidly oxidizes into its unreactive met form at acidic pH of 4.2, conducting these experiments using deoxygenated hemoglobin at a slightly higher pH of 5 or 6 may facilitate the oppor tunity to simultaneously capture a UV spectrum of NO bound hemoglobin from the reaction vessel, while observing the recovery of catalytic CO 2 from the MIMS instrument. In addition, experiments involving varied concentrations of NO and supplied dioxygen are necessary to unequivocally demonstrate that the time lag

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94 preceding the reversal of NO inhibition by abundant dioxygen is indeed due to an oxidation reaction on enzyme bound NO rather than simple displacement by O 2 Overall value of the nitric oxide studie s on OxDC is anchored on studying the reported dependence of catalysis on dioxygen and the search for the putative binding pocket for it on the enzyme. Earlier approaches toward these endeavors involved site directed mutagenesis on OxDC residues (e.g. Iso leucine 142), hypothesized to define the putative pocket for the dioxygen cofactor. R esidues of interest were identified from computer simulated models of the presumed M ichaelis M enten complex using the crystal structure as template The biggest h urdle in characterizing these enzyme constructs, especially from the perspective of oxygen dependence, was the set of limitations inherent to the FDH endpoint assay. Preliminary kinetic c h aracterization of site specific OxDC mutants on I142 have so far ind icated u nperturbed profiles of catalytic effects of oxygen Continued approaches or refinements of the MIMS method may be employed to characterize other targeted site specific mutants, or reaction conditions aimed at assessing the oxygen dependence of OxDC MIMS and Site specific M utants One of the main roadblocks previously mentioned in kinetically characterizing mutants of OxDC is having a mutant enzyme construct which is neither dead nor active to a considerable magnitude within the detection limits of t he FDH endpoint assay. The versatility of MIMS is not limited to the variety of buffer conditions one may perform with the method T he range of sensitivity of the instrument can be set in detecting even minute amounts of CO 2 generated in solution. This vit al feature revives the potential of earlier approaches in studying OxDC through site directed mutagenesis, render ing

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95 MIMS as a powerful tool in kinetically characterizing weakly active mutants demonstrating roles of other vital conserved residues. Effect o f Periodate and biSulfite anions Preliminary characteriz ation by MIMS, of anions with oxidizing and reducing properties have shown considerable magnitudes of inhibition with approximated Kis in the low millimolar range However a n interesting feature in t hese experiments was an apparent ly transient enhancement in catalytic generation of CO 2 prior to inhibition at high concentrations of inhibitor versus substrate. From these experiments one may speculate that the anion may be oxidizing/reducing the catalyti c Manganese to a state that is favorable for one step of catalysis, but rendering the enzyme unable to turnover. This may not be surprising since other manganese containing enzymes such as oxalate oxidase and Mn SOD, rely on a continuous toggle between two oxidation state s of manganese (Mn 2+ and Mn 3+ ) throughout their catalytic cycles. There has so far been no evidence for Mn 3+ in catalysis by wild type OxDC, although one cannot rule out the possibility of a rapidly transient (and yet undetected) existence of it during turnover. One may also argue that strong oxidizing agents may oxidize unreacted substrate and thus create artifactual generation of detected carbon dioxde. Regardless, it would be interesting to know whether anions c apable of redox chemistry can be instrumental in uncovering more information on the chemistry of the catalytic manganese of OxDC. P redictions on inhibitory molecules for OxDC have been patterned after precedents reported on other manganese containing enzymes of analogous metal env ironments in their ca talytic sites. H ow the chemistry of these e nzymes relate to one another remains a broad question motivating ongoing work in this project.

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96 APPENDIX A EXPRESSION AND PURIF ICATION OF BACILLUS SUBTILIS OXDC Comparative table of expre ssio n and purification schemes for untagged and polyhistidine tagged B. s ubtilis o xalate decarboxylase in BL21 (DE3) E.coli cells ( 10, 14, 18 ) Untagged Bs OxDC C terminally His 6 tagged Bs OxDC Yvrk gene/pET 9a plasm id construct Yvrk gene/pET 32 a plasmid construct EXPRESSION: Overnight cultures grown in Overnight cultures grown in 50mL LB with 100g Ampicillin Reinoculation of overnight cultures in: 3 Liters of LB and grown at 37 o C until Induction OD 600 = 0.6 1 L of LB at 37 o C and grown until Induction OD 600 = 0.3 Heat Shock at 42 o C for 10mins Heat Shock at 42 o C for 18mins Addition of: 1mM isopropyl D thiogalactopyranoside 5mM Manganese Chloride Grow for 4 hours at 30 o C Gro w for 4 hours at 37 o C Harvest cells and Lyse by Sonication (70% Amplitude) Overnight Extraction in : 50mM Imidazole Cl pH 7.0 1.0M NaCl 0.1% Triton X 100 10mM 2 mercaptoethanol PURIFICATION: Removal of cell debris by ce ntrifugation COLUMN 1 DEAE Sepharose (Anion Exchange) Nickel (Ni NTA) Affinity column Salting in 1.7M Ammonium Sulfate to precipitate out unwanted proteins COLUMN 2 Phenyl Sepharose (HIC) Concentrate eluted f raction pool and dialyze overnight into Storage buffer: 20mM Hexamethylenetetramine Cl 500mM NaCl pH 6.0 Buffer e xchange eluted fraction pool into Storage buffer: 50mM Tris Cl 500mM NaCl pH 8.5 Removal of adventitious metals Buffer exchange into Chel ex 100 buffer Treat buffer ex c hanged protein solution with Chelex 100 (BioRad) resin Concentrate metal free protein solution to 7 8mg/mL via YM 30 Centriprep (Millipore)

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97 Figure A 1 12% SDS PAGE gel of fractio ns from the expression and purification of C terminally His 6 tagged wild type Bs OxDC. Figure A 2 12% SDS PAGE gel of fractions from the expression and purification of untagged wild type Bs OxDC

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98 Figure A 3 12% SDS PAGE gel of fractions from the expres sion and purification of untagged C383S Bs OxDC mutant.

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99 APPENDIX B OXDC SEQUENCES AND A LIGNMENTS B.subtilis_OxDC -----------------------------------------------------------B.amyloliquefa sciens ----------------------------------------------------------A.niger_OxDC MQLTLPPRQLLLSFATVAALLDPSHGGPVPNEAYQQLLQIPASSPSIFFQDKPFTPDHRD 60 C.botulinum ---------------------------------------------MYIQN --------5 F.velutipes --MFNNFQRLLTVILLSGFTAG ----VPLASTTTGTGTATGTSTAAEPSATVPFAST D 52 B.subtilis_OxDC MKK -----QNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMKFSFS 53 B.amyloliquefasciens MKKLIQQLASKHLPQPIRGRKGATDEGPRNLARDFQNPDMLVPPSTDAGTVQNLKFSFS 59 Aniger_OxDC PYDHKVDAIGEGHEPLPWRMGDGATIMGPRNKDRERQNPDMLRPPSTDHGNMPNMRWSFA 120 C.botulinum QYQNLCNLLMSGCIPQPIRDGAGATDIGPRDILRDLENPDMLVPPSTDTGLIPNLKFSFS 65 F.velutipes PNPVLWNETSDPALVKPERNQLGATIQGPDNLPIDLQNPDLLAPPTTDHGFVGNAKWPFS 11 2 *** : : :***:* ** ** : ::.*: B.subtilis_OxDC DTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIREL H W H KEA E WAYMIYGSARVT 113 B.amyloliquefasciens DTHMRLEDGGWSREVTVRELPVSKNIAAVNMRLKPGAVREL H W H KEA E WGYVINGGVRLT 119 A.niger_OxDC DSHIRIEEGGWTRQTTVRELPTSKELAGVNMRLDEGVIREL H W H REA E WAYVLAGRVRVT 180 C.botulinum DTNMTIRPGGWSREITVRELPIATTMAGVNMRLTPGGVREV H W H QQS E WSYMLKGSARIT 125 F.velutipes FSKQRLQTGGWARQQNEVVLPLATNLACTNMRLEAGAIREL H W H KNA E WAYVLKGSTQIS 1 72 :: :. **::*: ** : :* .**** :**:***:::**.*:: .::: B.subtilis_OxDC IVDEKGRSFIDDVGEGDLWYFPSGLP H SIQAL --EEGAEFLLVFDDGSFS E NSTFQLTD 170 B.amyloliquefasciens AVDQNGRNFIDNVSEGDLWYFPSGIP H SIQGL --EQGSEFLLVFDDGSFSENSTFSVTD 17 6 A.niger_OxDC GLDLEGGSFIDDLEEGDLWYFPSGHP H SLQGLS -PNGTEFLLIFDDGNFSEESTFLLTD 238 C.botulinum AVDDRGRNFIADIGPGDLWFFPPLFP H SIQGL --EEGCEFLLLFDDGNFSDLRTFSLSE 182 F.velutipes AVDNEGRNYISTVGPGDLWYFPPGIP H SLQATADDPEGSEFILVFDSGAFNDDGTFLLTD 232 :* .* .:* : ****:**. ***:*. :* **:*:**.* *.: ** ::: B.subtilis_OxDC WLAHTPKEVIAANFGVT KEEISNLPGKEKYIFENQLPGSLKDDIVEGPNGEVPYPFTYR 229 B.amyloliquefasciens WFAHTPRSVLEANFGVS GYDLAYIHKKERYMFQLEPPPPIERAAVSSPEGTVLEPFSYK 2 35 A.niger_OxDC WIAHTPKSVLAGNFRMR PQTFKNIPPSEKYIFQGSVPDSIPKELPRN FKASKQRFTHK 296 C.botulinum FFAHYPKDVLAANFGVT KNCFNCLPEGQVYIYQDTIPGPLESEAIESPYGTIPQSYKHS 241 F.velutipes WLSHVPMEVILKNFRAKNPAAWSHIPAQQLYIFPSEPPADNQPDPVSP QGTVPLPYSFN 291 :::* .*: ** : : *:: :.. B.subtilis_OxDC LLEQEPIESEGGKVYIADSTNFKVSKTIASALVTVEPGAMREL H W H PNTHEWQYYISGKA 289 B.amyloliquefasciens LSRQEPLVTSGGRVKIVDSKTFKVSKTIAAALVEVEPGGMREL H W H PNTDEWQYYLSGEA 295 A.niger_OxDC MLAQEPEHTSGGEVRITDSSNFPISKTVAAAHLTINPGAIREM H W H PNADEWSYFKRGRA 356 C.botulinum LLAQKPMTTPGGSVRIADTSNFPVAKTTAAALVEIKPGGMREI H W H PN DEFQYFLTGQS 300 F.velutipes FSSVEPTQYSGGTAKIADSTTFNISVAIAVAEVTVEPGALREL H W H PTEDEWTFFISGN A 351 : :* ** *.*:..* :: : : ::**.:**:****. .*: :: *.: B.subtilis_OxDC RMTVFASDGHARTFNYQAGDVGYVPFAMG H YVENIG DEPLVFL E IFKDDHYADVSLNQW 348 B.amyloliquefasciens KMTVFAAEGRARTFNYQASDVGYVPIAMG H YVQNTG DTVLRFL E IFKSDRFEDVSLNQW 354 A.niger_OxDC RVTIFAAEGNARTFDYVAGDVGIVPRNMG H FIENLSDDEEVEVL E IFRADRFRDFSLFQW 416 C.botulinum RMTVFADTGASRTFDYRAGDVGYVPTGYG H YVQNIG NETVWFL E AFRSDRFKSISLSQM 359 F.velutipes RVTIFAAQSVASTFDYQGGDIAYVPASMG H YVENIG NTTLTYL E VFNTDRFADVSLS QW 410 ::*:** : **:* ..*:. ** **:::* : : ** *. *:: ..** B.subtilis_OxDC LAMLPETFVQAHLDLG KDFTDVLSKEKHPVVKKKCSK -----385 B.amyloliquefasciens LALTPQRFVEQTLNVS PAFARRLKKKKSPVVKWKHKQ -----391 A.niger_OxDC MGETP QRMVAEHVFKDDPDAAREFLKSVESGEKDPIRSPSE --457 C.botulinum MAITPQQLIESNLNVG PGFLNALSRSKFQCSVGPCFHKTECSD 402 F.velutipes LALTPPSVVQAHLNLDDETLAELKQFATKATVVGPVN ------447 :. .: : Figure B 1. Amino acid sequence alignment of several bacterial and fungal OxDC using ClustalW ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). Highlighted residues (orange) are conserved Mn(II) ligand bindin g residues; other conserved residues are indicated by an asterisk below each alignment set of rows.

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100 B.subtilis_OxDC -------------------------------------------------B.amyloliquefaciens_OxDC ------------------------------------------------A.niger_OxDC -MQLTLPPRQLLLSFATVA ----ALLDPSHGGPVPNEAYQQLLQIPA 42 C.subvermispora_OxOx MNEKLVSVFCAILVAISVSARPTGNDVFYLPRAVAVSSAGASSPASLSSG 50 B.subtilis_OxDC ---------------------MKK -----QNDIPQPIRGDKGATVKIP 22 B.amyloliquefaciens_OxDC ---------------------MKKLIQQLASKHLPQPIRGRKGATDEGP 28 A.niger_OxDC --SSPSIFFQDKPFTPDHRDPYDHKVDAIGEGHEPLPWRMGDGATIMGP 89 C. subvermispora_OxOx TESSSAAEPTETVPFASDDPNPRLWNIDT QDLSVVAPERGPLGAKIIGP 99 **. B.subtilis_OxDC RNIERDRQNPDMLVPPETDHGTVSNMKFSFSDTHNRLEKGGYAREVTVRE 72 B.amylol iquefaciens_OxDC RNLARDFQNPDMLVPPSTDAGTVQNLKFSFSDTHMRLEDGGWSREVTVRE 78 A.niger_OxDC RNKDRERQNPDMLRPPSTDHGNMPNMRWSFADSHIRIEEGGWTRQTTVRE 139 C.subvermispora_OxOx DNLPLDIQNADTLAPPTTDSGSIPNAKWPFALSHNTLYTGGWVRIQNNEV 149 : **.* ** ** *.: ::.*: :* : **: B.subtilis_OxDC LPISENLASVNMRLKPGAIRELHWHKEAEWAYMIYGSARVTIVDEKGRSF 122 B.amyloliquefaciens_OxDC LPVSKNIAAVNMRLKPGAVRELHWHKEAEWGYVINGGVRLTAVDQNGRNF 128 A.niger_OxDC LPTSKELAGVNMRLDEGVIRELHWHREAEWAYVLAGRVRVTGLDLEGGSF 189 C.subvermispora_OxOx LPIAKAMAGVNMRLEAGTIRELHWHNTPEWAYILKGTTQITAVDENGKNY 199 ** :: :*.*****. *.:******. .**.*:: .::* :* :* .: B.subtilis_OxDC IDDVGEGDLWYFPSGLPHSIQALE --EGAEFLLVFDDGSFSENSTFQLT 169 B.amyloliquefaciens_OxDC IDNVSEGDLWYFPSGIPHSIQGLE --QGSEFLLVFDDGSFSENSTFSVT 175 A.niger_OxDC IDDLEEGDLWYFPSGHPHSLQGLSP -NGTEFLLIFDDGNFSEESTFLLT 237 C.subvermispora_OxO x LANVGPGDLWYFPEGMPHSLQGTNASDEGSEFLLIFPDGTFDASNQFMIT 249 : :: *******.* ***:*. :*:****:* **.*. .. :* B.subtilis_OxDC DWLAHTPKEVIAANFGVTKEEISNLPGKEKYIFENQLPGSLKDDIVEGPN 219 B.amyloliquefaciens_OxD C DWFAHTPRSVLEANFGVSGYDLAYIHKKERYMFQLEPPPPIERAAVSSPE 225 A.niger_OxDC DWIAHTPKSVLAGNFRMRPQTFKNIPPSEKYIFQGSVPDSIPKELPRNFK 287 C.subvermispora_OxOx DWLAHTPKDVIAKNFGVDISEFDRLPSHDLYIFPGVAP PLDATAPEDPQ 298 **:****:.*: ** : : : : *:* .: : B.subtilis_OxDC GEVPYPFTYRLLEQEPIESEGGKVYIADSTNFKVSKTIASALVTVEPGAM 269 B.amyloliquefaciens_OxDC GTVLEPFSYKLSRQEPLVTSGGRVKIVDSKTFKVSKTIAAALVEVEPGGM 275 A.niger_OxDC ASKQR FTHKMLAQEPEHTSGGEVRITDSSNFPISKTVAAAHLTINPGAI 336 C.subvermispora_OxOx GTIPLPYSFEFSKVVPTQYAGGTVKIADTRTFPISKTISVAEITVEPGAM 348 ::..: ** *.*: .* :***:: : ::**.: B.subtilis_OxDC RE LHWHPNTHEWQYYISGKARMTVFASDGHARTFNYQAGDVGYVPFAMGH 319 B.amyloliquefaciens_OxDC RELHWHPNTDEWQYYLSGEAKMTVFAAEGRARTFNYQASDVGYVPIAMGH 325 A.niger_OxDC REMHWHPNADEWSYFKRGRARVTIFAAEGNARTFDYVAGDVGIVPRNMGH 386 C .subvermispora_OxOx REL HWHPTEDEWTFFIEGQARVTLFAGESNAQTYDYQGGDIAYIPTAYGH 398 **:****. .** :: *.*::*:**.:..*:*::* ..*:. :* ** B.subtilis_OxDC YVENIGD EPLVFLEIFKDDHYADVSLNQWLAMLPETFVQAHLDLG KDF 367 B.amyloliquefaciens_OxDC YVQNTGD TVLRFLEIFKSDRFEDVSLNQWLALTPQRFVEQTLNVS PAF 373 A.niger_OxDC FIENLSDDEEVEVLEIFRADRFRDFSLFQWMGETPQRMVAEHVFKDDPDA 436 C.subvermispora_OxOx YVENSGN TTLRFLEIFNSPLFQDVSLTQWLANTPRAIVKATLQLS DNV 446 :::* .: : .****. : *.** **:. *. :* : B.subtilis_OxDC TDVLSKEKHPVVKKKCSK --385 B.amyloliquefaciens_OxDC ARRLKKKKSPVVKWKHKQ --391 A.niger_OxDC AREFLKSVESGEKDPIRSPSE 457 C.subvermispora_OxOx IDSLNKSKAFVVAS D -----461 : *. Figure B 2 Amino acid sequence alignment of bacterial and fungal OxDC with the (blue) bicupin oxalate oxidase (OxOx) from yeast. Alignment was created using ClustalW 2.1 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ).

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101 (A) 1 MKKQNDIPQP IRGDKGATV K IPRNIERDRQ NPDMLVP PET DHGTVSNMKF SFSDT HNRLE 61 KGGYAREVTV RELPISENL A SVNMRLKPGA IRELHWH KEA EWAYMIYGSA RVTIV DEKGR 121 SFIDDVG EGD LWYFPSGLPH SIQAL EEGAE FLLVFDDGSF SEN STFQLTD WLAHTPKEVI 181 AANFGVTKEE ISNLPGKEK Y IFENQLPGSL KDDIVEG PNG EVPYPFTYRL LEQEP IESEG 241 GKVYIADSTN FKVSKTIAS A LVTVEPGAMR ELHWHPN THE WQYYISGKAR MTVFA SDGHA 301 RTFNYQAGDV GYVPFAMGH Y VENIGDEPLV FL EIFKDDHY ADVSLNQWLA MLPETFVQAH 361 LDLGKDFTDV LSKEKHPVV K KK C SK (B) 1 atgaaaaaac aaaatgacat tccgcagcca attagaggag acaaaggagc aacggtaaaa 61 atcccgcgca atattgaaag agaccggcaa aaccctgata tgctcgttcc gcctgaaacc 121 gatcatggca ccgtcagca a tatgaagttt tcattctctg atactcataa ccgattagaa 181 aaaggcggat atgcccggga agtgacagta cgtgaattgc cgatttcaga aaaccttgca 241 tccgtaaata tgcggctgaa gccaggcgcg attcgcgagc ttcactggca taaagaagct 301 gaatgggctt atatgattta cggaagtgca agagtcacaa ttgt agatga aaaagggcgc 361 agctttattg acgatgtagg tgaaggagac ctttggtact tcccgtcagg cctgccgcac 421 tccatccaag cgctggagga gggagctgag ttcctgctcg tgtttgacga tggatcattc 481 tctgaaaaca gcacgttcca gctgacagat tggctggccc acactccaaa agaagtcatt 541 gctgcgaact tcggcgtgac aaaagaagag atttccaatt tgcctggcaa agaaaaatat 601 atatttgaaa accaacttcc tggcagttta aaagatgata ttgtggaagg gccgaatggc 661 gaagtgcctt atccatttac ttaccgcctt cttgaacaag agccgatcga atctgaggga 721 ggaaaagtat acattgcaga ttcgac aaac ttcaaagtgt ctaaaaccat cgcatcagcg 781 ctcgtaacag tagaacccgg cgccatgaga gaactgcact ggcacccgaa tacccacgaa 841 tggcaatact acatctccgg taaagctaga atgaccgttt ttgcatctga cggccatgcc 901 agaacgttta attaccaagc cggtgatgtc ggatatgtac catttgcaat g ggtcattac 961 gttgaaaaca tcggggatga accgcttgtc tttttagaaa tcttcaaaga cgaccattat 1021 gctgatgtat ctttaaacca atggcttgcc atgcttcctg aaacatttgt tcaagcgcac 1081 cttgacttgg gcaaagactt tactgatgtg ctttcaaaag aaaagcaccc agtagtgaaa 1141 aagaaatg ca gtaaataa Figure B 3 (A) amino acid and (B) yvrk gene sequences of oxalate decarboxylase from B. subtilis. Highlighted residue in red (C383) is the only cysteine in the amino acid sequence.

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102 LIST OF REFERENCES 1. Emiliani, E., and B ekes, P. (1964) Enzymatic Oxalate Decarboxylation in Aspergillus Niger, Arch Biochem Biophys 105 488 493. 2. Shimazono, H. (1955) Oxalic Acid Decarboxylase, a New Enzyme from the Mycelium of Wood Destroying Fungi, J Biochem Tokyo 42 321 340. 3. Tanner, A ., and Bornemann, S. (2000) Bacillus subtilis YvrK is an acid induced oxalate decarboxylase, J Bacteriol 182 5271 5273. 4. Jonsson, S., Svedruzic, D., Wroclawska, E., Chang, C. H., and Richards, N. G. J. (2003) Structure and mechanism of enzymes mediating oxalate metabolism., Biochemistry 42 8617 8617. 5. Svedruzic, D., Jonsson, S., Toyota, C. G., Reinhardt, L. A., Ricagno, S., Lindqvist, Y., and Richards, N. G. J. (2005) The enzymes of oxalate metabolism: unexpected structures and mechanisms, Arch Bioche m Biophys 433 176 192. 6. Lane, B. G. (1994) Oxalate, germin, and the extracellular matrix of higher plants, FASEB J 8 294 301. 7. Tanner, A., Bowater, L., Fairhurst, S. A., and Bornemann, S. (2001) Oxalate decarboxylase requires manganese and dioxygen f or activity Overexpression and characterization of Bacillus subtilis YvrK and YoaN, J Biol Chem 276 43627 43634. 8. Woo, E. J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C., and Pickersgill, R. W. (2000) Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities, Nature Structural Biology 7 1036 1040. 9. Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 angs trom resolution, Biochem 41 7659 7669. 10. Just, V. J., Stevenson, C. E., Bowater, L., Tanner, A., Lawson, D. M., and Bornemann, S. (2004) A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site, J Biol Chem 279 19867 19874. 11. Dunwell, J. M. (1998) Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins, Biotechnol Genet Eng Rev 15 1 32. 12. Dunwell, J. M., Culham, A., Carter, C. E., Sosa Aguirre, C. R., and Goodenough, P. W. (2001) Evolution of functional diversity in the cupin superfamily, Trends Biochem Sci 26 740 746.

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103 13. Dunwell, J. M., Purvis, A., and Khuri, S. (2004) Cupins: the most functionally diverse protein superfamily ?, Phytochemistry 65 7 17. 14. Burrell, M. R., Just, V. J., Bowater, L., Fairhurst, S. A., Requena, L., Lawson, D. M., and Bornemann, S. (2007) Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282 ,000 by mutating an active site lid, Biochemistry 46 12327 12336. 15. Just, V. J., Burrell, M. R., Bowater, L., McRobbie, I., Stevenson, C. E., Lawson, D. M., and Bornemann, S. (2007) The identity of the active site of oxalate decarboxylase and the import ance of the stability of active site lid conformations, Biochem J 407 397 406. 16. Tabares, L. C., Gatjens, J., Hureau, C., Burrell, M. R., Bowater, L., Pecoraro, V. L., Bornemann, S., and Un, S. (2009) pH dependent structures of the manganese binding sit es in oxalate decarboxylase as revealed by high field electron paramagnetic resonance, J Phys Chem B 113 9016 9025. 17. Angerhofer, A., Moomaw, E. W., Garcia Rubio, I., Ozarowski, A., Krzystek, J., Weber, R. T., and Richards, N. G. (2007) Multifrequency E PR studies on the Mn(II) centers of oxalate decarboxylase, J Phys Chem B 111 5043 5046. 18. Moomaw, E. W., Angerhofer, A., Moussatche, P., Ozarowski, A., Garcia Rubio, I., and Richards, N. G. (2009) Metal dependence of oxalate decarboxylase activity, Bioc hemistry 48 6116 6125. 19. Dunwell, J. M., and Gane, P. J. (1998) Microbial relatives of seed storage proteins: conservation of motifs in a functionally diverse superfamily of enzymes, J Mol Evol 46 147 154. 20. Khuri, S., Bakker, F. T., and Dunwell, J. M. (2001) Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins, Mol Biol Evol 18 593 605. 21. Svedruzic, D., Liu, Y., Reinhardt, L. A., Wroclawska, E., Cleland, W. W., and Richards, N. G. (2007) Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis, Arch Biochem Biophys 464 36 47. 22. Chang, C. H., Richards, N. G. J. (2005) Intrinsic carbon carbon bond reactivity at the manganese cent er of oxalate decarboxylase from density theory, J. Chem. Theory Comput. 1 994 1007. 23. Reinhardt, L. A., Svedruzic, D., Chang, C. H., Cleland, W. W., and Richards, N. G. (2003) Heavy atom isotope effects on the reaction catalyzed by the oxalate decarbo xylase from Bacillus subtilis, J Am Chem Soc 125 1244 1252.

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104 24. Imaram, W., Saylor, B. T., Centonze, C. P., Richards, N. G. J., and Angerhofer, A. (2011) EPR spin trapping of an oxalate derived free radical in the oxalate decarboxylase reaction, Free Radi cal Biol. Med. 50 1009 1015. 25. Magro, P., Marciano, P., and Dilenna, P. (1988) Enzymatic Oxalate Decarboxylation in Isolates of Sclerotinia Sclerotiorum, Fems Microbiology Letters 49 49 52. 26. Schute, H., Flossdorf, J., Sahm, H., and Kula, M. R. (1976) Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii, European Journal of Biochemistry 62 151 160. 27. Muthusamy, M., Burrell, M. R., Thor neley, R. N., and Bornemann, S. (2006) Real time monitoring of the oxalate decarboxylase reaction and probing hydron exchange in the product, formate, using fourier transform infrared spectroscopy, Biochemistry 45 10667 10673. 28. Gross, J. H. (2006) Mass spectrometry : a textbook 1st ed., Springer, New York. 29. Kortz, L., Helmschrodt, C., and Ceglarek, U. (2011) Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids, Anal. Bioanal. Ch em. 399 2635 2644. 30. Matros, A., Kaspar, S., Witzel, K., and Mock, H. P. (2011) Recent progress in liquid chromatography based separation and label free quantitative plant proteomics, Phytochemistry (Elsevier) 72 963 974. 31. Hoch, G., and Kok, B. (196 3) A mass spectrometer inlet system for sampling gases dissolved in liquid phases, Arch Biochem Biophys 101 160 170. 32. Itada, N., and Forster, R. E. (1977) Carbonic anhydrase activity in intact red blood cells measured with 18O exchange, J Biol Chem 252 3881 3890. 33. Tu, C., Wynns, G. C., McMurray, R. E., and Silverman, D. N. (1978) CO2 kinetics in red cell suspensions measured by 18O exchange, J Biol Chem 253 8178 8184. 34. Brodbelt, J. S., Cooks, R. G., Tou, J. C., Kallos, G. J., and Dryzga, M. D. ( 1987) In vivo mass spectrometric determination of organic compounds in blood with a membrane probe, Anal Chem 59 454 458. 35. Silverman, D. N. (1982) Carbonic anhydrase: oxygen 18 exchange catalyzed by an enzyme with rate contributing proton transfer step s, Methods Enzymol 87 732 752. 36. Moral, M. E. G., Tu, C., Richards, N. G. J., and Silverman, D. N. (2011) Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases, Anal. Biochem. 418 73 77.

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105 37. Emiliani, E., and Riera, B. (1968) Enzymatic Oxalate Decarboxylation in Aspergillus Niger .2. Hydrogen Peroxide Formation and Other Characteristics of Oxalate Decarboxylase, Biochimica Et Biophysica Acta 167 414 &. 38. Delieu, T., and Walker, D. A. (1972) Improved Cathode for Me asurement of Photosynthetic Oxygen Evolution by Isolated Chloroplasts, New Phytologist 71 201 225. 39. Patil, P. V., and Ballou, D. P. (2000) The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments, Analytica l Biochemistry 286 187 192. 40. Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2000) Lehninger principles of biochemistry 3rd ed., Worth Publishers, New York. 41. Cornish Bowden, A. (1979) Fundamentals of enzyme kinetics Butterworths, London ; Boston. 42. Olivares, J. A. (1988) Inductively coupled plasma mass spectrmetry, Methods Enzymol 158 205 232. 43. Shimazono, H., and Hayaishi, O. (1957) Enzymatic decarboxylation of oxalic acid, J Biol Chem 227 151 159. 44. Tu, C., Swenson, E. R., and Silverman, D. N. (2007) Membrane inlet for mass spectrometric measurement of nitric oxide, Free Radic Biol Med 43 1453 1457. 45. Mikulski, R., Tu, C., Swenson, E. R., and Silverman, D. N. (2010) Reactions of nitrite in erythrocyte suspensions measured by membrane i nlet mass spectrometry, Free Radic Biol Med 48 325 331. 46. Hrabie, J. A., Klose, J. R., Wink, D. A., and Keefer, L. K. (1993) New Nitric Oxide Releasing Zwitterions Derived from Polyamines, Journal of Organic Chemistry 58 1472 1476. 47. Keefer, L. K., N ims, R. W., Davies, K. M., and Wink, D. A. (1996) "NONOates" (1 substituted diazen 1 ium 1,2 diolates) as nitric oxide donors: convenient nitric oxide dosage forms, Methods Enzymol 268 281 293. 48. Maragos, C. M., Morley, D., Wink, D. A., Dunams, T. M., S aavedra, J. E., Hoffman, A., Bove, A. A., Isaac, L., Hrabie, J. A., and Keefer, L. K. (1991) Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects, J Med Chem 34 3242 3247. 49. Hughes, M. N. (2008) Chemistry of nitric oxide and related species, Methods Enzymol. 436 3 19.

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106 50. Cleland, W. W. (1979) Statistical analysis of enzyme kinetic data, Methods Enzymol 62 151 160. 51. Wilkinson, G. N. (1961) Statistical estimations in enzyme kinetics, Biochem. J. 80 324 332. 52. Cleland, W. W. (1967) Statistical analysis of enzyme kinetic data, Adv. Enzymol. Relat. Subj. Biochem. 29 1 32. 53. Copeland, R. A. (2005) Evaluation of enzyme inhibitors in drug discovery : a guide for medicinal chemists and pharmacologists Wiley Interscience, Hoboken, N.J. 54. Moral, M. E. G., Tu, C. K., Imaram, W., Angerhofer, A., Silverman, D. N., and Richards, N. G. J. (2011) Nitric oxide reversibly inhibits Bacillus subtilis oxalate decarboxylase, Chemical Communication s 47 3111 3113. 55. Koppenol, W. H. (1998) The basic chemistry of nitrogen monoxide and peroxynitrite, Free Radic Biol Med 25 385 391. 56. Hess, D. T., Matsumoto, A., Kim, S. O., Marshall, H. E., and Stamler, J. S. (2005) Protein S nitrosylation: purview and parameters, Nat Rev Mol Cell Biol 6 150 166. 57. Reed, G. H., and Markham, G. D. (1984) EPR of Mn(II) Complexes with Enzymes and Other Proteins, in Biological Magnetic Resonance (Berliner, L. J., and Reuben, J., Ed.), pp 73 142, Plenum Press, New Yor k. 58. Hoffman Luca, C. G., Eroy Reveles, A. A., Alvarenga, J., and Mascharak, P. K. (2009) Syntheses, structures, and photochemistry of manganese nitrosyls derived from designed Schiff base ligands: potential NO donors that can be activated by near infrar ed light, Inorg Chem 48 9104 9111. 59. Eroy Reveles, A. A., Leung, Y., Beavers, C. M., Olmstead, M. M., and Mascharak, P. K. (2008) Near infrared light activated release of nitric oxide from designed photoactive manganese nitrosyls: strategy, design, and potential as NO donors, J Am Chem Soc 130 4447 4458. 60. Ford, P. C., Fernandez, B. O., and Lim, M. D. (2005) Mechanisms of reductive nitrosylation in iron and copper models relevant to biological systems, Chem Rev 105 2439 2455. 61. Chang, C. H., Svedru zic, D., Ozarowski, A., Walker, L., Yeagle, G., Britt, R. D., Angerhofer, A., and Richards, N. G. (2004) EPR spectroscopic characterization of the manganese center and a free radical in the oxalate decarboxylase reaction: identification of a tyrosyl radica l during turnover, J Biol Chem 279 52840 52849.

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107 62. Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems, Archives of Biochemistry and Biophysics 357 1 7 63. Borgstahl, G. E., Parge, H. E., Hickey, M. J., Beyer, W. F., Jr., Hallewell, R. A., and Tainer, J. A. (1992) The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4 helix bundles, Cell 71 107 118. 64. Lah, M. S., Dixon, M. M., Pattridge, K. A., Stallings, W. C., Fee, J. A., and Ludwig, M. L. (1995) Structure function in Escherichia coli iron superoxide dismutase: Comparisons with the manganese enzyme from Thermus thermophilus, Biochemistry 34 1646 1660. 65. Miller, A. F., Padmakumar, K., Sorkin, D. L., Karapetian, A., and Vance, C. K. (2003) Proton coupled electron transfer in Fe superoxide dismutase and Mn superoxide dismutase, J. Inorg. Biochem. 93 71 83. 66. Jackson, T. A., Karapetian, A., Miller, A. F., and Brunold, T. C. (2005) Probing the Geometric and Electronic Structures of the Low Temperature Azide Adduct and the Product Inhibited Form of Oxidized Manganese Superoxide Dismutase, Biochemistry 44 1504 1520. 67. Whittaker, J. W., and W hittaker, M. M. (1991) Active Site Spectral Studies on Manganese Superoxide Dismutase, Journal of the American Chemical Society 113 5528 5540. 68. Satyapal, and Pundir, C. S. (1993) Purification and properties of an oxalate oxidase from leaves of grain so rghum hybrid CSH 5, Biochimica Et Biophysica Acta 1161 1 5. 69. Purich, D. L. (2010) Enzyme Kinetics Catalysis and Control : A Reference of Theory and Best Practice methods Elsevier, Amsterdam.

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108 BIOGRAPHICAL SKETCH Mario Moral was born in Naples, Italy in 1971. He received his B S Chemistry degree in the Philippines in 1994 from the Ateneo de Manila University, where he was immediately invited to teach for the Department of Chemistry. In 1997 he entered th e religious order of the Society of Jesus and left the congregation upon completion of his novitiate formation. He worked for the Philippine Institute of Pure and Appl ied Chemi stry (PIPAC) in collaborative projects with the Philippine Research Institute while do ing graduate course work in molecular biology and biochemistry under the Department of Chemistry at the University of the Philippines in Diliman Mario started graduate studie s in the Department of Chemistry at the University of Florida in 2006 where he joined the res earch group of Nigel G. J. Richards. He graduated in December of 2011.