Chemical and kinetic characterization of glutamine hydrolysis catalyzed by Escherichia coli asparagine synthetase B

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Title:
Chemical and kinetic characterization of glutamine hydrolysis catalyzed by Escherichia coli asparagine synthetase B
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xiv, 165 leaves : ill. ; 29 cm.
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Schnizer, Holly G., 1969-
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Subjects / Keywords:
Glutamine -- chemistry   ( mesh )
Hydrolysis   ( mesh )
Aspartate-Ammonia Ligase -- metabolism   ( mesh )
Aspartate-Ammonia Ligase -- chemistry   ( mesh )
Aspartate-Ammonia Ligase -- physiology   ( mesh )
Catalysis   ( mesh )
Models, Chemical   ( mesh )
Papain -- metabolism   ( mesh )
Papain -- chemistry   ( mesh )
Papain -- physiology   ( mesh )
Escherichia coli -- chemistry   ( mesh )
Department of Biochemistry and Molecular Biology thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Biochemistry and Molecular Biology -- UF   ( mesh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1997.
Bibliography:
Includes bibliographical references (leaves 152-164).
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Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Holly G. Schnizer.

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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
        Page vii
    List of Figures
        Page viii
        Page ix
        Page x
        Page xi
    Abbreviations
        Page xii
    Abstract
        Page xiii
        Page xiv
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
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        Page 15
        Page 16
        Page 17
    Chapter 2. pH dependence of the glutaminase reaction catalyzed by asparagine synthetase B
        Page 18
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    Chapter 3. Effect of temperature on glutamine hydrolysis catalyzed by asparagine synthetase B
        Page 45
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        Page 47
        Page 48
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    Chapter 4. Determination of the rate determining step in asparagine synthetase B-catalyzed glutamine hydrolysis
        Page 67
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    Chapter 5. Evidence of a thioester intermediate formed during glutamine hydrolysis catalyzed by asparagine synthetase B
        Page 102
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    Chapter 6. Characterization of a thioester intermediate
        Page 120
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    Chapter 7. Discussion and future directions
        Page 138
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    References
        Page 152
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    Biographical sketch
        Page 165
        Page 166
        Page 167
Full Text









CHEMICAL AND KINETIC CHARACTERIZATION OF GLUTAMINE
HYDROLYSIS CATALYZED BY ESCHERICHIA COLI
ASPARAGINE SYNTHETASE B










BY

HOLLY G. SCHNIZER










A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA 1997























This work is dedicated to
my parents, Flint and Dottye Gray, and to my husband, Richard Schnizer,
with love.














ACKNOWvLEDGMENTS


I wish to thank everyone who helped me accomplish the work presented here. I would like to thank my supervisor, Dr. Sheldon Schuster, for his eagerness to discuss new ideas, his enthusiasm in my work, and for his friendship. My work benefited greatly from the guidance and helpful discussions provided by my supervisory committee: Dr. Tom O'Brien, Dr. Tom Yang, Dr. Richard Moyer, Dr. Harry Nick, and Dr. Don Allison. I would like to thank them especially for their continuing support even when my research took a change in direction. I am also indebted to Dr. Nigel Richards and Dr. Jon Stewart for their additional guidance in weekly discussions.I am especially grateful to my advisor on my first project on the hepatitis viruses, Dr. Johnson Lau, who introduced me to the fascinating world of viruses, and who had patience with me during my first few years in the laboratory.

My thanks goes to my friends in Dr. Schuster's Laboratory for providing the daily support I needed to complete this journey: Dr. Sue Boehlein, Ms. Ellen Walworth, and Mr. Gentry Wilkerson. I am especially thankful for Sue's helpful comments and ideas which helped guide my research.

Most of all I am grateful to my husband, Richard Schnizer, for all his helpful scientific advice and most of all for his love, patience, and support which helped me achieve this goal.


i1i














TABLE OF CONTENTS




PAGE

ACKNOW LEDGM ENTS ....................................................................................... iij

LIST OF TABLES .................................................................................................... vi

LIST OF FIGURES .................................................................................................. viii

ABBREVIATIONS .................................................................................................. xii

ABSTRACT ............................................................................................................... xiii

CHAPTERS

I INTRODUCTION ..................................................................................... 1

2 pH DEPENDENCE OF THE GLUTAMINASE REACTION 18
CATALYZED BY ASPARAGINE, SYNTHETASE B .................

Introduction ................................................................................... 18
M aterials and M ethods ............................................................. 23
Results .............................................................................................. 27
Discussion ....................................................................................... 40

3 EFFECT OF TEMPERATURE ON GLUTAMINE HYDROLYSIS 45
CATALYZED BY ASPARAGINE, SYNTHETASE B .................

Introduction ................................................................................... 45
M aterials and M ethods .............................................................. 46
Results .............................................................................................. 51
Discussion ....................................................................................... 64


iv







4 DETERMINATION OF THE RATE DETERMINING STEP
IN ASPARAGINE SYNTHETASE B-CATALYZED
GLUTAMINE HYDROLYSIS ........................................ 67

Introduction ............................................................ 67
Materials and Methods.............................................. 70
Results ..................................................................7
Discussion ............................................................... 98

5 EVIDENCE OF A THIOESTER INTERMEDIATE FORMED
DURING GLUTAMINE HYDROLYSIS CATALYZED BY
ASPARAGINE SYNTHETASE B...................................... 102


Introduction ............................................................ 102
Materials and Methods.............................................. 104
Results.................................................................... 106
Discussion ............................................................... 109

6 CHARACTERIZATION OF A THIOBSTER INTERMEDIATE ...120

Introduction............................................................ 120
Materials and Methods ............................................. 122
Results.................................................................... 123
Discussion............................................................... 134

7 DISCUSSION AND FUTURE DIRECTIONS ........................... 138

REFERENCES............................................................................. 152

BIOGRAPHICAL SKETCH ............................................................ 165













LIST OF TABLES


Table page

2.1 Oligonucleotides used in construction of sitedirected m utants of AS-B .................................................. 2 5

2.2 Kinetic constants of the AS-B-catalyzed hydrolysis
of L-glutam ine at 37C ......................................................... 3 1

2.3 Kinetic constants of the AS-B catalyzed hydrolysis
of L-glutamic-y-monohydroxamate at 370C ................3 4

2.4 Kinetic constants for glutamine hydrolysis
catalzyed by the AS-B mutant H47N at 37C ..............3 7

2.5 Values of pKa for the kcat/Km-pH profiles for
glutamine and LGH hydrolysis catalyzed by wildtype AS-B and for glutamine hydrolysis catalyzed
by the A S-B m utant H47N .................................................. 3 8

2.6 Kinetic parameters for wild-type AS-B and the
H29A, H80A, H47N, and R30A mutants at three
values of pH .............................................................................. 3 9

3.1 Thermodynamic properties of glutamine and LGH
hydrolysis catalyzed by wild-type AS-B and the
R 30A m utant ........................................................................... 5 6

3.2 Kinetic constants for the synthesis of LGH
catalyzed by wild-type AS-B ............................................. 5 9





vi







4.1 Kinetic constants for AS-B catalyzed hydrolysis of
L-glutamine, L-glutamic acid-7-monohydroxamate,
and L-glutamic acid-7-hydrazide ..................................... 7 8

5.1 Isolation of a covalent glutamyl-enzyme
interm ediate by gel filtration ........................................... 11 5

5.2 Time dependence of y-glutamyl AS-B adduct
degradation in SDS solution ................................................ 11 6

5.3 Trapping the thioester by filter binding ...................... 11 7

5.4 Base lability of the covalent adduct isolated by gel
filtration ..................................................................................... 11 9

6.1 Characterization of the glutamyl-enzyme
interm ediate ............................................................................ 1 2 9

7.1 Kinetic parameters at 5C for glutamine hydrolysis
catalyzed by wild-type AS-B according to schemes
(1) and (2) .................................................................................. 1 4 6

7.2 Kinetic parameters at 5C for glutamine hydrolysis
catalyzed by AS-B according to equation (3) ............. 149

7.3 Comparison of the actual and simulated values
obtained for the kcat, slope, intercept, and [TE]
from Figure 7.1 ........................................................................ 15 0















vii













LIST OF FIGURES

Figures age

1.1 Mechanism of nitrogen transfer via an ammonia
intermediate as proposed by Mei and Zalkin, 1989 1 0

1.2 Mechanism of nitrogen transfer via an imide
intermediate 3 as proposed by Richards and
Schuster, 1992 ....................................................................... 1 4

1.3 Mechanism of nitrogen transfer via direct transfer
of the nitrogen from glutamine to aspartyl-AMP
as proposed by Stoker et al., 1996 ................................. 1 5

2.1 Sequence alignment of part of the GAT-domains
of known asparagine synthetases and a partial set
of class II amidotransferases showing highly
conserved residues .............................................................. 2 2

2.2 The pH dependence of kcat for wild-type AS-B
catalyzed-glutamine hydrolysis ..................................... 2 9

2.3 The pH dependence of kcat/Km for wild-type AS-B
catalyzed glutamine hydrolysis ...................................... 3 0

2.4 The pH dependence of kcat for wild-type catalyzed
LG H hydrolysis ...................................................................... 3 2

2.5 The pH dependence of kct/Km for wild-type AS-B
catalyzed LGH hydrolysis ................................................... 33

2.6 The pH dependence of kcat for glutamine
hydrolysis catalzyed by the AS-B mutant H47N ......3 5



viii







2.7 The pH dependence of kcat/Km for glutamine
hydrolysis catalzyed by the AS-B mutant H47N ......3 6

3.1 Temperature dependence of glutamine hydrolysis
catalyzed by wild-type AS-B .......................................... 5 3

3.2 Temperature dependence of LGH hydrolysis
catalyzed by wild-type AS-B .......................................... 5 4

3.3 Temperature dependence of glutamine hydrolysis
catalyzed by the AS-B mutant R30A ............................ 55

3.4 Effect of hydroxylamine concentration on the
synthesis of LGH catalyzed by wild-type AS-B .........5 7

3.5 Effect of glutamate concentration on the synthesis
of LGH catalyzed by wild-type AS-B ............................5 8

3.6 Glutamate inhibition of the glutaminase reaction
catalyzed by wild-type AS-B at 5oC ............................... 60

3.7 Glutamate inhibition of the glutaminase reaction
catalyzed by wild-type AS-B at 37C ...........................6 1

3.8 Replot of the slope from the double-reciprocal
plots of glutamine hydrolysis at 5C versus
glutam ate concentration .................................................... 6 2

3.9 Replot of the slope from the double-reciprocal
plots of glutamine hydrolysis at 370C versus
glutam ate concentration .................................................... 6 3

4.1 Partitioning of the thioester intermediate to Lglutamic-y- monohydroxamate using
hydroxylamine in a reaction catalyzed by wt AS-B 7 6

4.2 Partitioning of the thioester intermediate to
glutamate hydroxamate using hydroxylamine in
the glutaminase reaction catalyzed by AS-B
R 30A ............................................................................................ 7 7


ix








4.3 DON inactivation of AS-B in the absence of
glutam ine .................................................................................. 8 1

4.4 Replot of DON inactivation of AS-B in the absence
(0) and presence of glutamine (A, 20 tM; 0,50
[IM ) ............................................................................................... 8 2

4.5 DON inactivation of AS-B in the presence of 20 j.M
glutam ine .................................................................................. 8 4

4.6 DON inactivation of AS-B in the presence of 50 jaM
glutam in e .................................................................................. 8 5

4.7 DON inactivation of AS-B catalyzed LGH hydrolysis
in the absence of LGH .......................................................... 8 6

4.8 Replot of DON inactivation of AS-B catalyzed LGH
hydrolysis ................................................................................. 8 7

4.9 The effect of various concentrations of LGH on
DON inactivation of AS-B catalyzed LGH hydrolysis 8 8

4.10 LGH protection against DON inactivation of AS-B
catalyzed LGH hydrolysis ..................................................8 9

4.11 DON inactivation of the R30A mutant of AS-B in
the absence of glutamine .................................................. 90

4.12 Replot of DON inactivation of R30A in the absence
of glutam ine ............................................................................ 9 1

4.13 Glutamine protection from DON inhibition of the
R30A catalyzed glutaminase reaction ........................... 92

4.14 Replot of glutamine protection of DON inactivation
of R30A catalyzed glutaminase ......................................9 3

4.15 Glutamate formation in AS-B catalyzed glutamine
hydrolysis as a function of time .....................................9 5


x









4.16 Effect of the presence of L-glutamate on the burst
in product formation in AS-B catalyzed glutamine
hydrolysis ................................................................................. 9 6

4.17 Mechanism of glutamine hydrolysis catalyzed by
the class II glutamine amidotransferases as
proposed by Mei and Zalkin (1989) .............................. 97

5.1 Effect of glutamine on the steady-state
concentration of the thioester .......................................... 11 8

6.1 Rate of deacylation of the thioester intermediate
formed during wild-type AS-B catalyzed
glutam ine hydrolysis ........................................................... 1 2 6

6.2 The steady-state concentration of the thioester
formed in the presence of ATP ........................................1 27

6.3 Effect of ATP on the rate of deacylation ......................1 28

6.4 Effect of ammonia on the rate of deacylation ...........1 3 0

6.5 Effect of glutamine on the steady-state
concentration of thioester formed in the
glutaminase reaction catalyzed by the AS-B
m utant, R 30A .......................................................................... 1 3 1

6.6 Effect of glutamine on the steady-state
concentration of the thioester formed during the
glutaminase reaction catalyzed by the AS-B
m utant, N 74A .......................................................................... 1 3 2

6.7 The rate of deacylation of the AS-B mutant, N74A,
during glutamine hydrolysis ............................................ 1 3 3

7.1 Comparison of the simulated to the actual data
representing glutamate formation as a function of
tim e .............................................................................................. 1 4 5


xi













ABBREVIATIONS ALL: Acute Lymphoblastic Leukemia AS: asparagine synthetase AS-A: ammonia dependent asparagine synthetase A from E.coli AS-B: asparagine synthetase B from E. coli AMP: adenosine monophosphate ATP: adenosine 5'-triphosphate CIA: mutant of AS-B with cysteine-I replaced by alanine DON: 6-diazo-5-oxonorleucine E. coli: Escherichia coli
GAT: glutamine amide transfer GFAT: glutamine fructose- 6-phosphate amidotransferase GPA: glutamine 5-phosphoribosyl-l-pyrophosphate
amidotransf erase
H47N: mutant of AS-B with histidine-47 replaced by asparagine KIE: kinetic isotope effect LGH: L-glutamic-7-monohydroxamate N74A: AS-B mutant with asparagine-74 replaced by alanine Ppi: pyrophosphate R30A: AS-B mutant with arginine-30 replaced by alanine wt: wild-type







XH













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHEMICAL AND KINETIC CHARACTERIZATION OF GLUTAMINE HYDROLYSIS CATALYZED BY ESCHERICHIA COLI ASPARAGINE SYNTHETASE B

By

Holly Schnizer

December, 1997

Chairman: Dr. Sheldon Schuster Major Department: Biochemistry and Molecular Biology

A detailed model of glutamine hydrolysis catalyzed by
Escherichia coli asparagine synthetase B, a class II amidotransferase, was constructed using a variety of chemical and kinetic techniques. Glutamine hydrolysis catalyzed by the class II amidotransferase family has been proposed to follow a mechanism similar to that of amide hydrolysis catalyzed by the thiol protease, papain. This study presents a comparison of the catalytic behavior of these two hydrolytic enzymes in order to determine if asparagine synthetase Bcatalyzed glutaminase reaction can be described by a similar mechanism. First, this study revealed differences in acid-base catalysis between these two hydrolytic enzymes. In a continuing investigation of the importance of conserved histidines in the


xiii








glutamine utilizing domain of asparagine synthetase B, the similarity of pH profiles of wild-type and an asparagine synthetase B mutant in which a conserved histidine is replaced with an asparagine, suggests that this histidine is not likely to be involved as part of the cys-his dyad which is essential to papain-catalyzed hydrolysis. Second, this study describes the development of a model for glutamine hydrolysis catalyzed by wild-type asparagine synthetase B from a minimal mechanism. The use of such a model was confirmed by the demonstration that one main feature of papain catalysis, the formation of a thioester, was conserved in asparagine synthetase B catalyzed glutamine hydrolysis.
The values for the dissociation constants for glutamine and the inhibition constants for glutamate were determined. Furthermore, the isolation of a putative thioester intermediate provided the opportunity to measure the rate of a single step in the reaction, deacylation. These constants along with the information gained from the kinetic constants of L-glutamic-y-monohydroxamate synthesis catalyzed by asparagine synthetase B were used in a computer simulation of the burst of glutamate production with time which showed that an additional rate limiting conformational change must be included to describe the observed kinetic behavior of wild-type asparagine synthetase B.









xiv












CHAPTER 1
INTRODUCTION



L-Asparagine and Treatment of Malignant Disease
L-asparagine was one of the first amino acids to be isolated from nature (Vauquelin & Robiquet, 1806). The importance of this non-essential amino acid in such metabolic events as the production of aspartate and ammonia (Broome, 1963a), transamination (Meister, 1952), protein synthesis (Cooney & Handschumacher, 1970), and glycosylation (Spiro, 1969) has been established. Considerable interest in L-asparagine metabolism developed after the 1953 discovery (Kidd, 1953a, 1953b) of the therapeutic effects of guinea pig serum against lymphomas in mice; and the subsequent identification of L-asparaginase, the enzyme which catalyzes asparagine hydrolysis, as the inhibitory component (Broome, 1961, 1963a, 1963b). Today, L-asparaginase is used most frequently in the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children. When L-asparaginase is used as the sole chemotherapeutic agent, 40-68% of the patients achieve complete remission (Capizzi, et al., 1971; Uren & Handschumacher, 1977). In combination with other chemotherapeutic agents, L-asparaginase treatment results in almost 95% remission in previously untreated ALL (Sanz, et al., 1986). Despite the therapeutic potency of Lasparaginase, this treatment has several major disadvantages which




2


limit its use. First, L-asparaginase is toxic to organs such as the liver, pancreas, kidneys, blood clotting systems, and brain therefore, therapy is often accompanied by a wide variety of deleterious side effects (Crowther, 1971). These side effects are usually reversible and dose dependent however, in some cases they preclude the use of this drug. A second drawback to the use of L-asparaginase is that some patients who achieve remission suffer relapse with tumors resistant to the L-asparaginase therapy (Kiriyama et al., 1989; Lobel et al., 1979; Terebello et al., 1986). And finally, L-asparaginase treatment is confined to ALL because the enzyme may actually enhance the growth of some resistant tumors (Capizzi et al., 1971; Tallal et al., 1970) and, at present, there is no assay to predict sensitivity in solid tumors.
The therapeutic action of L-asparaginase is related to its ability to deplete the extracellular supply of L-asparagine (Broome, 1961; Uren & Handschumacher, 1977). While normal cells are independent of an external source of asparagine, sensitive tumor cells synthesize asparagine very slowly and are dependent on extracellular supplies of this amino acid to survive. Indeed, it has been shown that the activity of the enzyme which synthesizes asparagine, asparagine synthetase (AS), is depressed in tumor cells which are sensitive to asparaginase (Hutson et al., 1997). In contrast, increased AS activity is often observed in cell lines which exhibit a resistance to Lasparaginase (Gantt & Arfin, 1981; Kiriyama et al., 1989; Patterson & Orr, 1969) and a direct correlation between increased AS mRNA and protein content and resistance to asparaginase has been demonstrated in a human leukemia cell line (Hutson et al., 1997).




3


In view of the above mentioned problems associated with
asparaginase therapy, alternative methods of treating ALL need to be explored. Since the effectiveness of this therapy seems to reside in reducing the circulating concentration of asparagine, inhibitors of asparagine synthetase could provide this much needed alternative. In efforts to achieve this goal, several hundred compounds have been evaluated as AS inhibitors, however none have provided both the strength and specificity required for clinical consideration (Cooney et al, 1976). Therefore, a detailed chemical and kinetic investigation of AS is essential in order to develop more effective inhibitors of this enzyme.

Asparagine Synthetase
Asparagine synthetases catalyze the ATP dependent transfer of nitrogen from either glutamine or ammonia to aspartic acid in the formation of asparagine. The biochemical and mechanistic properties of AS have been examined in a variety of sources including mammals (Hongo et al., 1978; Markin et al., 1979; Markin et al., 1981; Milman & Cooney, 1979; Van Heeke & Schuster, 1989b), fungi (Ramos & Wiame, 1979, 1980), plants (Pike & Beevers, 1982; Rognes, 1970), and bacteria (Burchall et al., 1964; Cedar & Schwartz, 1969a, 1969b; MacPhee et al., 1983) as well as in some tumor cell lines (Gantt & Arfin, 1981). In order to facilitate the recovery of sufficient amounts of AS for study, expression systems have been developed for the overexpression of human AS in yeast (Van Heeke & Schuster, 1990) and AS-B in Escherichia coli (Boehlein et al., 1994a).




4


Two classes of AS have been described which do not possess
any sequence similarity. AsnA, found in many procaryotes, encodes for an AS (AS-A) which only uses ammonia as a source of nitrogen in the synthesis of asparagine (reaction 2). The kinetic and chemical mechanism of AS-A, the product of the asnA gene, has been well characterized (Cedar & Schwartz, 1969a, 1969b). In fact, the existence of an aspartyl-AMP intermediate formed during AS catalyzed asparagine synthesis was demonstrated first in experiments using AS-A (Cedar & Schwartz, 1969b). The other AS, encoded by asnB found in both prokaryotes and eukaryotes, catalyzes the synthesis of asparagine using either ammonia or glutamnine as a source of nitrogen (reactions 1 and 2). These enzymes also catalyze glutamine hydrolysis (reaction 3) in the absence of the other substrates; a reaction which is stimulated by the presence of ATP.


L-Asp + L-Gln + ATP L-Asn + L-Glu +AMP + PP (Reaction 1) L-Asp + NH3 + ATP 4 L-Asn + AMP + PP, (Reaction 2)
L-Gln + H,O L-Glu + NH, (Reaction 3)


Glutamine dependent asparagine synthetases are thought to be organized into two functional domains. The glutamine amide transfer (GAT) domain binds glutamine and catalyzes the hydrolysis of glutamine to glutamate and ammonia. The synthetase domain binds ATP and aspartate and catalyzes the ammonia dependent reaction as well as the transfer of nitrogen to aspartate in the glutamine dependent reaction. Evidence for the location of the two




5


active sites in separate domains was revealed through studies using monoclonal antibodies (Pfeiffer et al., 1986, 1987) and chemical modifiers (Larsen & Schuster, 1992) that could selectively inhibit the glutaminase or asparagine synthesis activities of human or bovine AS.
While the mechanism of nitrogen transfer between the two domains is poorly understood, examination of conserved residues within the individual domains has led to the identification of binding sites and intermediates involved in the reaction. For the C-terminal synthetase domain, it has been confirmed that during asparagine synthesis, a P-aspartyl-AMP intermediate is formed which activates the aspartate carboxylate for attack by the amide nitrogen of glutamine (Luehr & Schuster, 1985). Further studies showed a binding site for aspartate located in a region highly conserved among asparagine synthetases (Boehlein et al., 1997a). And finally, another highly conserved region in the AS synthetase domain was found to contain a P-loop-like motif that is present in other enzymes which catalyze the hydrolysis of ATP to AMP and PP (Bork & Koonin, 1994), and has been proposed to represent a pyrophosphate binding element (Boehlein et al. manuscript in progress).
Experiments using site-directed mutagenesis have identified several conserved amino acids in the N-terminal domain that are crucial to glutamine utilization. Sequence analysis reveals that all AS contain an N-terminal cysteine residue. Milman and Cooney first proposed a critical cysteine residue in the glutamine active site when they found AS from mouse pancreas was strongly susceptible to oxidation (Milman & Cooney, 1979). More evidence for an important




6


cysteine residue was revealed upon the observation that bovine pancreatic AS lost glutamine dependent activity when it was covalently modified with the sulfhydryl modifier and glutamine analog, 6-diazo-5-oxo-L-norleucine (DON) (Mehlhaff & Schuster, 1991). Finally, the essentiality of the N-terminal cysteine residue was demonstrated in human AS and E. coli AS-B in experiments replacing cysteine-1 with either an alanine or serine residue (Boehlein et al., 1994a; Sheng et al., 1993; Van Heeke & Schuster, 1989b).
In addition to the conserved cysteine residue, the role of
another completely conserved residue, asparagine-74, also has been determined. When Asn-74 was replaced with either an alanine or an aspartate, the resulting mutant enzymes (N74A & N74D) exhibited a depressed glutaminase activity (Boehlein et al., 1994b). Subsequent studies using the alternative substrate, L-glutamic acid-'ymonohydroxamate (LGH) and the AS-B mutant N74A led to the suggestion that the interaction of this asparagine residue with substrates causes a destabilization in the ES complex (Boehlein et al., 1996) and, therefore, this residue might play a role similar to that of Gln-19 of papain (Drenth et al., 1976; Menard, 1995). For papain, it is thought that Gln-19 forms an 'oxyanion hole' in the stabilization of a tetrahedral intermediate formed when the active site cysteine attacks the substrate. This 'oxyanion hole' contains a hydrogen bonding network involving the NH peptide backbone of cys-25 and the side chain NH, of Gln-19 which stabilizes the oxide anion bonded to the tetrahedral carbon atom. The proposed role of N74 of AS-B in forming such an oxyanion hole is illustrated below:




7


O
1 ENZ
ASN-74 NH HN


H2N- C- S

R
CYS-1



This proposed relationship between the thiol protease, papain, and AS-B was confirmed in studies which showed that a nitrile could act as a weak competitive inhibitor of wild type AS-B and as a substrate for the AS-B mutant N74D (Boehlein et al., 1997b); results which were consistent with similar studies using wild type and a Q19E mutant of papain (Menard, 1995).


Glutamine Amidotransferase Class II
Based on its primary amino acid sequence, asparagine synthetase is a member of a family of enzymes known as the glutamine-dependent amidotransferases. The members of this family catalyze the transfer of nitrogen from glutamine to a variety of acceptor substrates in the biosynthesis of amino acids, carbohydrates, and nucleotides (for review, Zalkin, 1993). Like AS, all amidotransferases share a modular organization with a glutamine utilizing (GAT) domain in common and a synthetase domain which is unique to each member of the family. According to the amino acid sequence alignment of the GAT domain, glutamine amidotransferases are divided into two types (Zalkin, 1993). Class I enzymes (formerly 'G-type' for the trpG gene product, anthranilate synthase) are




8


characterized by three catalytically important residues thought to function as a cys-his-glu catalytic triad analogous to the thiol proteases. This class of enzymes (see Zalkin, 1993 for review) includes GMP synthetase (Tiedeman et al, 1985), anthranilate synthase, (Nichols et al., 1980) carbamoyl-phosphate synthetase (Piette, 1984), imidazole glycerol-phosphate synthase (Carlomagno, 1988), aminodeoxychorismate synthase, CTP synthetase (Weng, 1986), and FGAM synthetase (Schendel et al., 1989). Asparagine synthetase as well as glutamine 5'-phosphoribosyl-1-pyrophosphate amidotransferase (GPA) (Tso et al., 1982), glutamine fructose-6phosphate amidotransferase (GFAT) (Badet-Denisot et al., 1993), and glutamate synthase (Vanoni et al., 1991) comprise the class II amidotransferases (formerly 'F-type' for the purF gene product, glutamine 5-phosphoribosylpyrophosphate) which are characterized by a conserved N-terminal cysteine required for glutamine dependent activity.


Proposals for the Mechanism of Nitrogen Transfer
For the class II enzymes, the formation of a covalent yglutamyl-enzyme intermediate involving the N-terminal cysteine residue was first suggested in studies of GPA where it was shown that attachment of glutamine affinity analogs to cys-1 (Vollmer et al., 1983) or replacement with phenylalanine (Mantsala & Zalkin, 1984) caused selective elimination of the glutamine-dependent activity. Later studies using site directed mutagenesis of conserved residues in the GAT domain of GPA led to the proposal that cys-1 participated as part of a cys-his catalytic dyad in a mechanism analogous to the




9


thiol proteases (figure 1.1)(Mei and Zalkin, 1989). This model of nitrogen transfer involves a two step process: hydrolysis of glutamine to produce free ammonia, followed by its transfer to the acceptor substrate. In analogy to the thiol protease mechanism, a nucleophilic attack of the conserved cys-1 thiolate anion on the amide of glutamine results in a tetrahedral intermediate which, upon release of ammonia, is converted to an acylthioenzyme intermediate

1. The released ammonia is transferred to the activated aspartate intermediate 2 in the synthetase domain for the synthesis of asparagine while the acylenzyme intermediate undergoes hydrolysis to glutamate and free enzyme via a second tetrahedral intermediate. For AS, this model for nitrogen transfer is supported by the observation that all members of its family can catalyze the synthesis of asparagine using ammonia instead of glutamine, demonstrating the presence of binding site for ammonia. Furthermore, a completely conserved cysteine has been shown to be catalytically essential in GPA (Mei & Zalkin, 1989) and AS-B (Boehlein et al., 1994a) and a residue (Asn-74) thought to play a role in forming an oxyanion hole, much like that formed by Gln-19 in papain catalysis (Menard et al. 1995), has been shown for AS-B.
While there is some evidence supporting an ammonia mediated nitrogen transfer, there are also many inconsistencies. First, was the inability to show that the class II enzymes contain a catalytically important histidine cognate to that of the thiol protease papain. Papain requires an active site histidine which functions to stabilize the cysteine thiolate anion and provide a proton to the leaving group in the conversion of the first tetrahedral intermediate to the





10




( O NH2
H NH2 H HCys H

H O I
HisCYs O O HS102
2 H (N C02- <\ I

Cys1 0'

Asp2 + NH3 NH3


0
S
H3N K N
N' Cys / His12
CO2 N
O H


Asp29


(b) O O
Ad O
O 'O 0 NH
0- H NH2
H,- ),jk + AMP
H3N' C2O H
HO OH H3N "CO2
2

(C) 0 0

H3 H o H
HcCys / Hise2 + H20 H3N' N Hiso2
His10 + H20 4, H <
CO2 N C02 S N'r
O H H' transfer CysH

Asp29O Asp29




Figure 1.1 Mechanism of nitrogen transfer via an ammonia intermediate as proposed by Mei and Zalkin, 1989. (a) Mechanism for the hydrolysis of L-glutamine to yield ammonia and an acylenzyme intermediate 1 by analogy to the purF enzyme, GPA. (b) Synthesis of L-asparagine by reaction of ammonia with activated aspartyl-AMP 2. (c) Hydrolysis of the acylenzyme intermediate to yield L-glutamate. This diagram was taken from Richards and Schuster, 1992.








thioester. Initially, it was thought, from results of site-directed mutagenesis (Mei & Zalkin, 1989), that His-102 was involved in such acid-base catalysis for GPA and covalent modification studies using diethyl pyrocarbonate suggested that GFAT (Badet-Denisot & Badet, 1992) also possessed this important histidine. However, sitedirected mutagenesis of each of the conserved histidines in the GAT domain of AS-B revealed little or no change in the kinetic parameters (Boehlein et al., 1994a). Subsequent structural studies of GPA (Kim et al., 1996; Smith et al., 1994) and the N-terminal domain of GFAT (Isupov et al., 1996) did not reveal a histidine in the appropriate position to participate in acid-base catalysis. Alternatively, these structural studies led to the proposal that the a-amino group of cys-1 serves as the general acid-base catalyst (Smith, 1995). Subsequent structural comparisons of GPA with other hydrolytic enzymes placed the class II enzymes into a larger superfamily called the N-terminal nucleophile (Ntn) hydrolases which includes the 20S proteasome and penicillin acylase (Brannigan et al., 1995; Duggleby et al., 1995; Seemuller et al., 1996). This family is characterized by an N-terminal catalytic nucleophile which is thought to be activated by proton transfer from its side chain to the free N-terminus (Seemuller et al., 1996). However, experiments aimed at establishing the functional role of the N-terminal amine using mutants of GPA and GFAT with an additional N-terminal residue have yielded contradictory results (Isupov et al., 1996; Kim et al., 1996).
Further evidence against an ammonia mediated nitrogen
transfer in AS-B catalyzed asparagine synthesis was revealed in an investigation of the kinetic isotope effects (KIE) (Stoker et al. 1996)




12


for AS-B catalyzed glutamine dependent reactions. The KIEs associated with placing 5N in the primary amide of glutamine were examined for both glutamine dependent reactions and the resulting values were interpreted using 5N KIE determinations for papain catalyzed peptide hydrolysis (O'Leary et al, 1.974). For papain, the KIE value was shown to be consistent with a mechanism where the first irreversible step is C-N bond cleavage to form the thioester intermediate (O'Leary et al., 1974). It was expected that C-N bond cleavage would be rate limiting in the AS-B catalyzed glutaminase reaction as well and thus give the same 15N (V/K) isotope effect as papain. Furthermore, an ammonia mediated nitrogen transfer would require a common pathway for glutamine hydrolysis in the two glutamine dependent reactions. Therefore, if AS-B followed an ammonia mediated mechanism, it was expected that the 15N KIEs determined for both glutamine dependent reactions would be identical. Surprisingly, the glutaminase 15N (V/K) was significantly smaller than that obtained for papain catalyzed peptide hydrolysis, indicating that a step other than C-N bond cleavage may be rate limiting. Moreover, the KIE for glutaminase was smaller than that for the glutamine dependent synthesis reaction. These results were regarded as the first direct evidence against the mediation of nitrogen transfer by enzyme-bound ammonia.
In addition to the above mentioned inconsistencies between
AS-B catalytic behavior and an ammonia mediated nitrogen transfer, questions addressing the difficulties of maintaining the free ammonia in the active site in its unprotonated form led to the proposal of alternative mechanisms for nitrogen transfer (Figure 1.2) (Richards &




13


Schuster, 1992). The first proposal for an alternate mechanism of nitrogen transfer involved the formation of an imide intermediate (Richards & Schuster, 1992). According to this hypothesis, a direct nucleophilic attack of the primary amide of glutamine on the Paspartyl-AMP intermediate 2 to form an imide intermediate 3. Reaction of this intermediate with cys-1 causes the release of asparagine and the formation of a thioacylenzyme intermediate 1 which forms free enzyme and glutamate in a mechanism similar to the ammonia mediated mechanism. An attempt to isolate such an intermediate was made using the AS-B mutant (CIA) which lacks the cysteine required to catalyze C-N bond cleavage of the imide intermediate. However, an imide intermediate was not detected by HPLC using chemically synthesized imide as a standard, when CiA AS-B was incubated with glutamine, ATP, and aspartic acid. It was concluded that participation of an imide intermediate in nitrogen transfer catalyzed by AS-B was unlikely (Boehlein et al., unpublished observations).
In a second alternative hypothesis for the mechanism of

nitrogen transfer (Stoker et al., 1996), Cys-1 reacts with glutamine to form the first tetrahedral intermediate 5 (figure 1.3). In contrast to the ammonia mediated mechanism, however, the tetrahedral intermediate makes a subsequent direct attack on the P-aspartylAMP intermediate to form an intermediate in which glutamine is covalently linked to f3-aspartyl-AMP 6. Cleavage of the C-N bond in this intermediate releases the acylenzyme and forms a second tetrahedral intermediate 8. Asparagine is then formed from a loss of





14











oc oo o o o con"

NH 0, 0 H'transter N N*H
HN NHlj 0 AMP HH
H H3N 0 02 HO OH 3 H H
CO2 sH 2 CO2- HO


Cys, SO 1 O
sCysi Asps,
Asp3y


0 00
1 S HN
H S Asn H3N Cys, H C '
H3N K,, H3N CO"
Cys0 + H20 CO2 1 0
COz O. H'O


Asp3 Aspz
H' transfer
-H'

0


H3N O"

CO2 2SH
cys, 0

Asp33









Figure 1.2 Mechanism of nitrogen transfer via an imide intermediate
3 as proposed by Richards and Schuster, 1992. Attack of the primary amide occurs directly on the activated L-aspartate 2. Cys-1 and Asp-33 are then involved in the hydrolysis of the imide to yield L-glutamate and L-asparagine in subsequent steps.






15
















0 CYG,4 0NMO 0
M~j- H +H 1 H

CH, 0-7 HOO0
S' o*






0' 0' 0* N 0 H, H IH.N i CO;



H





H H




0







H2NN'













Figure 1 .3 Mechanism of nitrogen transfer via direct transfer of the nitrogen from glutamine to aspartyl-AMP as proposed by Stoker et al., 1996).




16


AMP from the tetrahedral intermediate. Interestingly, this mechanism would not necessarily require N-protonation before C-N bond cleavage in the glutamine dependent synthesis reaction and, therefore, may explain the differences in the 15N (V/K) isotope effects for the two glutamine dependent reactions.
Despite the progress that has been made in understanding the functions of the individual domains of AS, the mechanism of nitrogen transfer remains unequivocally defined. One difference between the mechanism involving free ammonia and that of a direct attack is the requirement, in the former, of a common mechanism for glutamine hydrolysis in the glutaminase and glutamine dependent asparagine synthesis reactions. Therefore, it is important to understand the mechanistic details of both the glutamine dependent synthesis and glutamine hydrolysis in order to discriminate between the two mechanisms. The ability of AS-B to catalyze the hydrolysis of glutamine in the absence of the other amino acids provides the opportunity to develop a model of the glutaminase reaction from which to identify the individual steps and intermediates in the reaction as well as examine the functional roles played by the conserved amino acids in the GAT domain. In efforts to begin building such a model, this dissertation focuses on the chemical and kinetic mechanisms of glutamine hydrolysis catalyzed by AS-B. Differences and similarities in the mechanism of substrate hydrolysis catalyzed by AS-B and papain will be examined through pH analysis of AS-B catalyzed glutaminase activity and by isolation of a covalent intermediate in the reaction pathway of glutamine hydrolysis catalyzed by AS-B. A variety of kinetic techniques will be used to




17


determine the individual rate constants of the glutaminase reaction and these will be used to build a model of glutamine hydrolysis catalyzed by AS-B.













CHAPTER 2
pH DEPENDENCE OF THE GLUTAMINASE REACTION CATALYZED BY ASPARAGINE SYNTHETASE B


Introduction
Asparagine Synthetase B (AS-B) catalyzes the ATP dependent synthesis of asparagine using aspartic acid and either glutamine or ammonia as a nitrogen source (reactions 1 and 2). In the absence of aspartate and ATP, this enzyme also is capable of catalyzing glutamine hydrolysis (reaction 3).


L-Gln + ATP + L-Asp -- L-Asn + AMP + PP + L-Glu (reaction 1) NH3 + ATP + L-Asp -- L-Asn + AMP + PPi (reaction 2)
L-Gln + H,20 --> Glu + NH3 (reaction 3)


Amino acid sequence alignment of the N-terminal glutamine utilizing domain of AS-B indicates that this enzyme is a member of the class II glutamine amidotransferase family (Zalkin, 1993) which includes glutamine 5'-phosphoribosylpyrophosphate amidotransferase (GPA) (Tso et al., 1982), glutamine fructose-6-phosphate amidotransferase (GFAT) (Badet-Denisot & Badet, 1993), and glutamate synthase (Vanoni et al., 1991). Like AS-B, members of this family of enzymes catalyze the transfer of the amide nitrogen of glutamine to various acceptor molecules including amino acids, nucleotides, and carbohydrates.


18




19


The mechanism of nitrogen transfer remains controversial and poorly understood. Results from studies using site-directed mutagenesis suggested the catalytic importance of a conserved N-terminal cysteine, a histidine, and an aspartic acid in glutamine utilization in GPA (Mei & Zalkin, 1989). This observation led to the hypothesis that glutamine amidotransferases catalyze glutamine hydrolysis by a mechanism similar to that of amide hydrolysis catalyzed by the thiol proteases, typified by papain (for review see Brocklehurst et al., 1987), as shown below (1).


Binding Acylation Deacylation
0 0NH, HHO
R-C R- C HS-Enz -' -R- CO aR- C + HS-Enz (1)
NH, NH2 O. S-Enz O OH
+I
R-C"NH2 R-C-OH
HS-Enz
S-Enz S-Enz
TH I TH II


In analogy to the papain mechanism, a nucleophilic attack of the Nterminal cysteine on the side-chain amide of glutamine forms a tetrahedral intermediate (TH I). Protonation of the amide allows C-N bond cleavage to produce a thioester intermediate and free ammonia. The anunmonia is then transferred to the acceptor molecule and the thioester intermediate is broken down, via another tetrahedral intermediate (TH II), to produce glutamate and free enzyme. As illustrated above, an active site cysteine and an acid-base catalyst (a role played by his-159 in papain) are vital components to such a reaction scheme. The cysteine is involved in the activation of glutamine as described above. In papain, the histidine is thought to




20


play three essential acid-base roles including 1) stabilization of the thiolate; 2) donation of a proton to the amide of the first tetrahedral intermediate to increase its leaving group ability; and finally 3) activation of water for a nucleophilic attack on the thioester intermediate by accepting a proton (Storer & Menard, 1994).
This mechanism of ammonia mediated nitrogen transfer is
supported by the ability of the amidotransferases to use ammonia as a nitrogen source and thus the demonstration of an ammonia binding site. Furthermore, the presence of a completely conserved Nterminal cysteine, which has been shown to be essential for glutamine dependent activity of GPA (Mei & Zalkin, 1989) as well as both human and E. coli asparagine synthetases (Boehlein et al., 1994a; Sheng et al., 1993; Van Heeke & Schuster, 1989b), suggests that this residue may function in a manner similar to that of the active site cysteine of the thiol proteases. On the other hand, the presence of an important histidine in the active site of the class II enzymes has become increasingly doubtful. While the studies of GPA mentioned above (Mei & Zalkin, 1989) and chemical modification studies of GFAT (Badet-Denisot & Badet, 1992) seemed to indicate the presence of an essential histidine in these enzymes, site-directed mutagenesis of residues, known to be conserved among asparagine synthetases, did not reveal any histidines required for activity in ASB (Boehlein, et al., 1994a). Moreover, recent structural studies of GPA (Kim et al., 1996; Smith et al., 1994) and the glutamine utilizing domain of GFAT (Isupov et al., 1996) suggested that there were no histidines in position to act as part of a cys-his dyad. Structural comparisons later revealed that the protein fold of GPA (Brannigan et




21


al., 1995) was homologous to that of a family of N-terminal nucleophile (Ntn) hydrolases which also includes penicillin acylase (Duggleby et al., 1995), the 20S proteasome (Lowe et al., 1995; Seemuller et al., 1996), and aspartylglucosamidase(Fisher et al., 1993; Oinonen et al. 1995). It is thought that members of the Ntn family use the N-terminal Ser, Thr, or Cys as the nucleophilic catalyst and the N-terminal c-amino group as the acid-base catalyst. However, whether or not the class II amidotransferases use the Nterminal amino group in acid-base catalysis remains uncertain as results from experiments which have placed an extra amino acid at the N-terminus of GPA (Kim et al., 1996) have been inconclusive.
Further investigation is necessary in order to understand fully how acid-base catalysis occurs during the glutamine hydrolysis catalyzed by the class II amidotransferases. In fact for AS-B, the possibility of an essential histidine still remains. A computer model of a hypothetical AS-B glutamine binding site, constructed using the crystallographic coordinates for the covalent adduct of DON and E. coli GPA, revealed that histidine-47 of AS-B was in an appropriate position to function as part of a cys-his dyad cognate to that of papain (Richards & Schuster, 1997). Moreover, this histidine is partially conserved in asparagine synthetases and a histidine in a comparable position is found in several other class II amidotransferases (Figure 2.1), thus its importance cannot be overlooked. In an effort to gain a clearer understanding as to how acid-base catalysis occurs during glutamine utilization by AS-B, it is the purpose of this chapter to explore the glutaminase activity of this





22





LtHMAS CGIWAL< >HRGPD< >FGFHRLAV< >NGEIYNHKAL GnHmAS CGIWAI,< >HRGPD< >FGFHRLAV< >NGEIYNEKAL RatAS CGIWAL< >HRGPD< >FGFHRLAV< >NGEIYNEKAL
MurAS CGIWAL< >HRGPD< >FGFHRLAV< >NGE-YNHKAL
HuriAS CGIWAL< >HRGPD< >FGFHRLAV< >NGEIYNHKKM SoyAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEEL
LotAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIFNHEEL
FavaAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEEL PeaNAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEEL AlflAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEDL PeaRAS CGILAV< >HRGPE< >LAQQRLAI< >NGEIYNHEDL AspaAS CGILAV< >HRGPD< >LSHQRLAI< >NGEIYNHEEL ArabAS CGILAV< >HRGPD< >LAHQRLP.V< >NGEIY.HEEL BrssAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEEL RiceAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNKEEL MaizAS CGILAV< >HRGPD< >LAHQRLAI< >NGEIYNHEEL ScerAS CGIFAA< >HRGPD< -FVHERLAI< >NGEIYNHIQL CeleAS CGVFSI< >HRQPD< >LVHERLAI< >NGEIVNHGEL ECoAS CSIFQV< >HRQPD< >LAHERLSI< >NQEIYNHQAL
1 29 47 74 80
ScerGA CGILGI< >HRGQD< >FTQQRVS.< >NGNLVNTASL HumGS CGIFAY< >YRGYD< >HKQQDMDL< >NGIITNYKDL
ScerGS CGIFGY< >YRGYD< >.TKQNPNR< >NGIITNFREL






Figure 2.1: Sequence alignment of part of the GAT-domains of known asparagine synthetases and a partial set of class II amidotransferases showing highly conserved residues. Numbering is according to the amino acid sequence of AS-B. Sequence. alignments taken from Richards & Schuster, 1997. LtHmAS = Cricetulus longicaudatus AS; GnHmAS = Mesocricetus auratus AS; RatAS = Rattus norvegicus AS; MurAS = Mus musculus AS; HumAS = Homo sapiens AS; Soy AS = Glycine max AS; Lot AS = Lotus japoni cus AS; Fava AS Vicia jaba AS; PeaNAS Pisum sath a AS (root); AspaAS = Asparagus officials AS; ArabAS Arabidopsis thaliana AS; BrssAS = Brassica oleracea AS; RiceAS = Oryza sativa AS; MaizAS = Zea ma vs; Scer AS Saccharomyces cerevisiae AS; CeleAS = elegant AS; EcoAS = Escherichia coli AS, ScerGA Saccharomyces cerevisiae GPA; HumGS Homo sapiens GFAT-, ScerGS Saccharomyces cerevisiae GFAT.




23


enzyme as a function of pH. In order to address the possibility that H47functions in an acid-base capacity, the effects of mutations at this position on the pH-rate profile will be characterized.


Materials and Methods
Chemicals and Reagents
Restriction and modifying enzymes were purchased from Promega (Madison, WI) or New England Biolabs (Beverly, MA). Other chemicals, including L-glutamine and L-glutamic-y-monohydroxamate, were obtained from SIGMA Chemical Company (St. Louis, MO) and were of the highest commercial purity. Oligonucleotide primers were synthesized on an Applied Biosystems 380B DNA synthesizer by the DNA Synthesis Core Facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Polymerase chain reactions (PCR) were performed on an Ericomp (San Diego, CA) thermocycler using the GeneAmp DNA Amplification Reagent Kit with AmpliTaq from PerkinElmer. Double stranded DNA sequencing was performed using the U.S. Biochemical Corp. Sequenase 2.0 Sequencing kit. Bacterial Strains and Plasmids
Bacterial strains BL21DE3pLysS (F, ompT, rb, mb-), supplied by Studier and Moffat; and NM522 (supE, thi (lac-proAB), hsd5, (rm)/F' pro AB, lac Iq Z M15), obtained from Stratagene (La Jolla, CA), are both derivatives of Escherchia coli K-12. The plasmid pBluescript was obtained from Stratagene (La Jolla, CA) and the plasmid pETB has been described previously (Boehlein et al., 1994a). E. coli host cells were transformed according to the procedures of Hanahan (Hanahan, 1983).




24


Enzyme Preparation

Construction of recombinant wild-type AS-B and mutants in
which His-29 or His-80 have been replaced by alanine (H29A, H80A) has been described elsewhere (Boehlein et al., 1994a). PCR cassette mutagenesis was used to replace Histidine-47 with alanine (H47A) or asparagine (H47N) using oligonucleotide primer pairs ss35OR and ss65 or ss348R and ss65, respectively (Table 2-1) and pETB as a template. The PCR parameters involved 35 cycles of denaturation at 94C for 1 min; annealing at 550C for 1 min; and extension at 72C for two minutes and the final cycle was followed by a 5 minute extension step at 72C. The plasmid, pETB, and the PCR products were digested with HpaI and XhoI and the 50 bp product was cloned into pETB. The resulting plasmids were sequenced through the insertion and no alterations other than those intended were found. All wild-type and mutant proteins were expressed, and purified using standard procedures (Boehlein et al., 1994a). Protein concentrations were determined by the method of Bradford (1976) using an assay kit supplied by Bio-Rad and mouse immunoglobulin G to construct a standard curve. During the course of these investigations an alternate method of determining the protein concentration revealed that the current use of mouse immunoglobulin G as a standard gives erroneously high protein concentrations for AS-B. Amino acid composition analysis of AS-B, carried out by the Protein Chemistry Core Facility of ICBR at the University of Florida, showed that the actual value for the concentration of this enzyme is 2.7 times less than that obtained using the Bradford assay. Therefore, all of the values of enzyme




25


concentration and therefore all kcat and kcat/Km values given in this chapter have been corrected by dividing the protein concentration by the factor 2.7.


TABLE 2.1
Oligonucleotides used in construction of site-directed mutants of AS-B


Oligo Oligonucleotide Sequence
Number

ss 6 5 5' A GCT TCC CAT ATG TGT TCA ATT TTT GGC GTA
TTC GAT 3'
ss350R 5' CGC CCC CGC GTT' AAC GTC GAC AAT TGA CAG ACG
TTC GGC GGC GAG A 3'
ss349R 5' CGC CCC CGC GTT AAC GTC GAC AAT TGA CAG ACG
TTC GGC GGC GAG A 3'

Glutaminase Assays
Initial rates of L-glutamine and L-glutamic-y-monohydroxamate (LGH) hydrolysis were determined by a modified procedure which uses glutamate dehydrogenase in the presence of NAD+ to measure glutamate concentration (Bernt and Bergmeyer, 1974). Reactions (100 p1) containing 100mM Bis-Tris and Tris-HC1 (pH 6.0-9.0) each (the kinetic constants appear to be independent of the nature and concentrations of these buffers), 8mM MgCl2 and various concentrations of glutamine or LGH were initiated by the addition of wild-type (0.7-2 gg) or mutant (4 pg) AS-B and incubated at 370C. A control containing denatured enzyme was used to monitor any glutamate formed from spontaneous breakdown. The reactions were terminated by boiling and a coupling reagent (300 mM glycine, 250




26


mM hydrazine, pH 9, 1 mM ADP, 1.6 mM NAD+ 2.2 units of glutamate dehydrogenase) was added and incubated 10 minutes at room temperature. The absorbance at 340 nm was measured and the concentration of glutamate was determined by comparison to a standard curve. Initial velocities were measured at 9 different substrate concentrations and each velocity was an average of 2-4 measurements. Stability of the wild-type and mutant AS-B enzymes at each pH was confirmed by incubation in buffer of the appropriate pH for 20 minutes followed by an assay of activity at pH 8. Initial velocity conditions were confirmed at each pH by linear plots of v versus time and v versus enzyme concentration. Data Analysis
The values of Km and kcat were determined from least-squares fit to vo vs [S] plots using the Prism software package supplied by Graphpad, Inc., (San Diego, CA) according to equation 1:



V max IS]
Km + IS]



Where v is the maximal velocity, [S] is the concentration of st Vmax is the maximal velocity, and Km is the Michaelis constant. The pH dependence of kcat/Km obtained from equation 1 were fit to the bell-shaped curve described by equation 2:



Ylim
Y = (2)
1 + 10(pkl-pH) + 10(PH-PK2)




27


In the above equation, Y is the observed kca/Km at a given pH, Yjj is the maximum value of kcat/Km, and pK1 and pK. are the lower and higher acid dissociation constants, respectively.


Results
Effect of pH on the Steady-state Kinetic Parameters of wild-type ASB Catalyzed Glutaminase

The steady-state kinetic parameters, kcat and kat/Km, for glutamine hydrolysis were measured as a function of pH. As illustrated in Figures 2.2 and 2.3, and Table 2.2, kcat is constant over the pH range 6.0-9.0 whereas the kcat/Kmexhibits a bell shaped curve. Since the pKa of the c-amino group of glutamine is 9.13, the Km and thus k1at/KM were corrected for protonated glutamine using the Henderson-Hasselbach equation in an attempt to show how the ionization of the substrate might affect the kcat/Km-pH profile. Indeed, the correction caused a complete disappearance of the basic limb in the kcat/K.(app) plot (Figure 2.3). Non-linear regression of the experimental data to equation 1 allowed determination of the pKa for the non-corrected data which is shown in Table 2.5.
A comparison of the pH dependencies of the hydrolyses of
glutamine and the alternate substrate, LGH, can provide information about whether these reactions follow the same mechanism. Therefore, a pH profile of the wild-type AS-B catalyzed LGH hydrolysis was also examined (figure 2.4 and 2.5, and Table 2.3). Similar to the glutamine profile, the kcat appeared independent of pH for most of the pH range studied with slight increases at the low and high pH. The kcar/Km,(app) profile for LGH is bell shaped with a peak




28


at neutral pH and an acidic and basic pKa close to that obtained with glutamine (Table 2.5). Unlike the glutamine profile, correction for protonated LGH had little effect on the pH-kcat/Km profile (Figure 2.5). Kinetic Characterization of the Glutaminase Reaction catalyzed by the AS-B mutant H47N
As part of a continuing search for a histidine which may serve in a general acid-base capacity during AS-B catalyzed glutamine hydrolysis, Histidine-47 was examined using site-directed mutagenesis. Mutations were made which replaced the histidine with an alanine (H47A) or an asparagine (H47N) and the resulting enzymes were expressed. H47A was insoluble whereas H47N expressed at high levels and was successfully purified. The glutaminase activity of H47N was approximately 10-fold slower than that of wild-type AS-B (compare Tables 2.2 and 2.4). In addition to these changes in kct, there was a slight increase in the Km value for glutamine. pH Dependence of the Kinetic Constants of wild-type AS-B and its H47N
The pH dependence of the kinetic constants for H47N catalyzed glutamine hydrolysis was examined. The pH dependence of kcac was minimal (figure 2.6) except for a slight decrease at low pH which was not evident in the wild-type. The pH profile of kcat/Km(app) revealed a bell-shaped curve (figure 2.7) similar to that of wild-type catalyzed LGH hydrolysis except the curve was slightly narrower. The decrease in kcat/Km(app) on the basic limb was evident even after the correction for protonated glutamine.




29








3.0

2.5-

2.0
a
1.5


0.5

0.0- I I I I I
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH


Figure 2.2 The pH dependence of kct for wild-type AS-B catalyzed glutamine hydrolysis. The kinetic constants were determined as described in materials and methods.





30










3000


2500


2000

1500E

1000


500
A
0I I I I I I
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 pH










Figure 2.3 The pH dependence of keat /Km for wild-type AS-B catalyzed glutamine hydrolysis. The kinetic constants were determined as described in the materials and methods. The solid line (-) represents the actual data. The dashed line (---) represents the data corrected for the protonation of glutamine.




31













Table 2.2
Kinetic constants of the AS-B-catalyzed hydrolysis of L-Glutan-ine at 37C


pH kcat K. kcat/Km
(sec') (mM) (Msec-')

6.00 1.51 0.05 5.78 0.60 261 6.25 1.67 0.10 3.44 0.33 485 6.50 1.89 0.03 2.4 0.1 787
7.00 1.94 0.03 1.01 0.06 1921 7.25 2.05 0.05 1.22 0.08 1680 7.50 1.92 0.03 1.01 0.06 1901 7.75 2.13 0.03 1.32 0.07 1614 8.00 2.21 0.03 1.15 0.05 1922 8.50 2.46 0.05 1.50 0.06 1640 8.75 2.48 0.05 1.92 0.09 1292 9.00 2.56 0.05 2.33 0.12 1099




32












5











0



5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH


Figure 2.4 The pH dependence of k,:a, for wild-type catalyzed LGH hydrolysis. The kinetic constants were determined as described in materials and methods.





33














5000



4000



3000

E
2000



1000


0I I I I I I
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 pH







Figure 2.5 The pH dependence of kcat/Km for wild-type AS-B catalyzed LGH hydrolysis. The kinetic constants were determined as described in materials and methods. The solid line (-) represents the actual data. The dashed line (---) represents the data corrected for the protonation of glutamine.




34












Table 2.3:
Kinetic constants of the AS-B-catalyzed hydrolysis of Lglutamic-y-monohydroxamate at 37C


pH kcat at k nKm
(sec') (mM) (M-'sec-')

6.00 3.46 0.05 6.51 0.29 531 6.25 3.16 0.05 2.39 0.15 1322 6.50 2.86 0.03 1.11 0.05 2577 6.75 2.59 0.03 0.68 0.02 3809 7.00 2.67 0.03 0.66 0.02 4045 7.25 2.65 0.03 0.58 0.03 4569 7.50 2.67 0.03 0.65 0.02 4108 7.75 2.65 0.03 0.63 0.03 4206 8.00 2.38 0.05 0.83 0.05 2867 8.25 2.54 0.03 1.11 0.05 2288 8.50 3.00 0.05 1.53 0.08 1961 8.75 3.10 0.05 2.26 0.11 1372 9.00 4.05 0.19 7.41 0.78 546




35










0.6

0.5
f
,,p.. 0.4%%o 0.3- m

0.2-

0.1

0.0.
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH


Figure 2.6 The pH dependence of k,,at for glutamine hydrolysis catalyzed by the AS-B mutant H47N. The kinetic constants were determined as described in materials and methods.




36












200



150



100
5


50



0 I I ,I I
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
pH











Figure 2.7 The pH dependence of kat/Km for glutamine hydrolysis catalyzed by the AS-B mutant H47N. The kinetic constants were determined as described in materials and methods.




37
















Table 2.4:
Kinetic constants for glutamine hydrolysis catalyzed
by the AS-B mutant H47N at 370C

pH kcat Km kcat/Km

(sec-') (mM) (M-'sec-')

6.00 0.19 0.01 18.6 2.8 10
6.25 0.22 0.008 13.1 2.0 17
6.50 0.30 + 0.005 8.3 0.9 36
6.75 0.30 0.008 5.4 0.4 55
7.00 0.30 0.005 3.2 0.2 94
7.25 0.30 0.003 2.4 0.1 125 7.50 0.38 + 0.005 2.5 0.1 152 7.75 0.32 0.003 2.5 0.1 128 8.00 0.32 0.003 3.0 0.1 107 8.25 0.40 0.005 4.1 0.2 98
8.50 0.46 0.008 6.1 0.3 75
8.75 0.38 0.01 7.8 0.7 49
9.00 0.43 0.01 10.5 1.1 41




38
















Table 2.5:
Values of pKa for the kat/Km-pH profiles for glutamine and LGH
hydrolysis catalyzed by wild-type AS-B and for glutamine hydrolysis
catalyzed by the AS-B mutant H47N


Enzyme Substrate km,/Km
pKI pK,
wild type glutamine 6.7 0.1 8.8 0.1
LGH 6.7 0.1 8.0 0.1

H47N glutamine 7.2 0.2 8.1 0.2






39

















cn +I +I





+1 +1 +1 mt +1
I-C c C Ln cl W-) M
Ln C'I 09 t:
ct C-i C,4 Ln








00


+1 +1 +1 +1
CL4 00
C:) cc
E




+1 +1 +1 +1
M M Ln
3u C14 OC 00







00 cn





+1 +1
r- cn C
In +1
cq
Lr



+1 +1 +1 Ln

cz 00 -Z m





N oc




40


Comparison of the Kinetic Parameters of Wild-type, H80A. and H29A, at various pH values
The kinetic constants for the glutaminase activity of the three mutants (H29A, H47N, and H80A) are compared with wild-type in Table 2.6. Substitution of an alanine for the histidines at positions 29 and 80 has little effect on the kcat at pH 6.5 and 7.5 while this substitution decreases kcat/Km significantly. However, unlike wild type the k,,t value of H80A increases with increasing pH to a value at pH 9 which is double that of wild type. The most striking effect caused by the mutations at positions 29 and 80 is seen in the dramatic increase in Km especially at high pH.


Discussion
The importance of a histidine in the mechanism of glutamine hydrolysis catalyzed by AS-B and thus the mechanistic similarity between the AS-B catalyzed glutaminase reaction and the papain catalyzed peptide hydrolysis have been questioned for several years. Papain catalyzes the hydrolysis of peptide, amide, and ester bonds by the mechanism shown in (1). All substrates tested with papain have shown a bell-shaped pH dependence for kcat/Km with pKa's of approximately 4 and 8.5. As shown in the simplified model below, it is considered that these ionizations represent the thiol of cysteine-25 and the imidazole of histidine-159 which are involved in the acylation process (Menard et al., 1990):




41


pK1 4 pK2 8.5
-SH +HIm- -S- +Him- -S- Imactive enzyme
form



In order to understand how acid-base catalysis occurs in the AS-B active site during glutamine hydrolysis, the pH dependence of the kinetic constants has been examined for this enzyme. For both glutamine and LGH, the kt/Km value has a bell-shaped pH dependency. In contrast, kct appears independent of pH in the range of 6-9. The similarity in pH profiles for these two substrates suggests that their hydrolysis occurs by similar mechanisms. Furthermore, the values of kcat/Km for LGH are about 2-fold higher than those for glutamine (compare Tables 2.2 and 2.3). If substrate hydrolysis follows a mechanism like that of papain, then Nprotonation of the leaving group on the first tetrahedral intermediate is required for C-N bond cleavage to occur. Therefore, the faster acylation rate for LGH, reflected in kcat/KM, is consistent with the fact that hydroxylamine (pKa=8.2) is a better leaving group than ammonia (pKa-9.2). The pH independence of kcat may arise from a change in rate determining step between two steps with similar rates. However, further investigation, using a more extensive pH range is necessary for a clearer picture of the ionizations affecting kcat. Unfortunately, the AS is highly unstable when the pH is greater than

9 or less than 6.
The bell-shaped curves displayed in the kcat/Km profiles for ASB hydrolysis of glutamine and LGH (with pKa values of 6.6 and 8.3) are narrower than that reported for papain. The stability of the ion-




42


pair in papain is reflected in the width of the pH-profile for kc t/K. (Menard et al., 1991). Therefore, it is possible that AS-B contains an ion pair like that of papain but lacks some of the factors which play a role in ion pair stabilization. On the other hand, the pH profile of kcat/Km may relate to ionizations in free substrate. In this study, the pKa values of the c-amino groups of the substrates are 9.13 therefore, the possibility that the alkaline pKa values observed in the kCat/Km profiles arise from the ionization of glutamine and LGH cannot be excluded. In fact, the basic limb of the glutamine kcat/Km profile is completely eliminated when Km values are corrected for protonated glutamine. However, the same treatment has little effect on the ktKm profile for LGH.
While the above examination of the kinetic behavior of wildtype AS-B as a function of pH indicates the potentiality of an ion pair similar to that of papain, past efforts have failed to reveal a histidine which functions as part of that pair. In this study, substituting an asparagine for histidine at position 47 resulted in a reduction of both kcat and kcat/Km of approximately 10-fold for the glutaminase activity. While these changes are significant, a much more dramatic effect was expected if this residue were playing such an essential role in catalysis as has been postulated. For example, replacement of histidine-353 with an asparagine in the class I enzyme, CPS, resulted in a drop in glutaminase activity from 1.1 ptmol/homg for wt to <0.006 itmol/hemg for H353N (Miran et al, 1991). Subsequent structural studies which showed that this residue was in position to interact with the active site histidine added further support that His353 was involved in acid-base catalysis (Thoden et al., 1997).




43



Additionally, the pH profiles for glutamine hydrolysis catalyzed by H47N (Figures 2.6 & 2.7) resemble those of wt including similar pKa values of 7.2 and 8.5 (Table 2.5) which suggests that histidine-47 is not responsible for one of these ionizations.

Previous investigations of H29 and H80 failed to reveal that a histidine in either of these positions was essential for AS-B catalyzed asparagine synthesis (Boehlein et al., 1994a). While these studies were performed at pH 8 in the presence of an imidizole buffer, it is possible that the importance of the active site histidine could have been masked. Table 2.6 presents the results of a further investigation using the glutaminase activity at pH 6.5, 7.5 and 9.0. The lack of a significant decrease in the k at value for the glutaminase reaction catalyzed by these mutants confirms the previous conclusions that these histidines probably are not involved as part of the essential cys-his dyad which has been proposed.
Interpretations of this and any pH analysis of an enzymatic reaction rely on a number of assumptions which have been summarized by Knowles (1976). First, as noted by Peller and Alberty (1959), the association of the pKa values of the pH-kca,/Km with ionizations of the free enzyme and free substrate assumes that only one ionization state of the enzyme is directly convertible to product. In fact, the pH dependence of the kcat/Km values observed in papain catalyzed substrate hydrolysis has been found to be more complex than originally thought (Brocklehurst, 1994). Several studies have provided evidence that the development of the full nucleophilic character of the ion pair depends not only on the ionization at pH 4 but on a second ionization at pH 4 and, in some cases, ionization with





44


pKa 5 (Brocklehurst, 1988 a,b). Altered pKa values also may result if the rate constant for substrate dissociation is less than the net rate constant for product formation. This is unlikely to be the case for AS-B glutamine hydrolysis since investigations into the dissociation constant of glutamine have suggested that a rapid equilibrium mechanism is plausible (see chapter 4). Determination of the pKa of a competitive inhibitor or of the backwards reaction will be necessary to confirm this assumption.
With respect to these assumptions it is important to take heed in comparison of the pH profiles of AS-B and papain. On the other hand, with the results presented here, it is difficult to envision a role for histidine-47 in acid-base catalysis when one considers the lack of effect its replacement with asparagine made on the pH profiles. In light of the presence of this histidine in many asparagine synthetases as well as other class II amidotransferases it was important to include this residue among potential acid-base catalysts. Indeed, the 10-fold loss of activity in the H47N mutant may reflect a minor or indirect importance of this residue in glutamine binding and/or utilization. On the other hand, the importance of the histidine to papain activity is underscored in the complete conservation of this amino acid across the entire family of cysteine proteases. The lack of such a completely conserved histidine in the class II family of amidotransferases suggests that an alternate mechanism of acid-base catalysis probably occurs in these enzymes and therefore demonstrates dissimilarities in mechanism for the two families of enzymes.














CHAPTER 3
EFFECT OF TEM~vPERATURE ON GLUTAMINE HYDROLYSIS CATALYZED BY ASPARAGINE SYNTHETASE B


Intro du ct ion

The following chapters present experiments which are aimed at the determination of rate constants and the identification of intermediates in order to develop a descriptive model of the glutaminase reaction. Much of the investigation requires a very slow reaction rate and, therefore, the reactions were examined at 5'C. Variation in temperature can cause alterations in the mechanism of a reaction including differences in rate determining steps. In order to validate the comparison of the results from experiments at 5'C with those performed at the temperature at which the reaction is normally investigated (37C), the steady state kinetics of glutamine hydrolysis will be examined over a range of temperatures and Arrhenius plots will be used to detect any changes in rate limiting step with temperature for this reaction. The temperature dependence of LGII hydrolysis catalyzed by wild-type AS-B and that of glutamine hydrolysis catalyzed by the AS-B mutant R30A will be examined in a similar manner and the activation energies of the three reactions will be compared. Similarity in the pH profiles for glutamine and LGH hiydrolysis presented in the previous chapter suggests that these reactions occur by a common mechanism. Further evidence for a common mechanism could be obtained 45




46



through similarities in the activation parameters which would suggest that the same step is rate limiting in both reactions. In contrast, work in the following chapters suggests that glutamine hydrolysis catalyzed by the AS-B mutant, R30A, has a different rate limiting step than that catalyzed by wt AS-B and these differences might be revealed in different values for the activation parameters.
A complete description of the glutaminase reaction requires 1) the order of product release, 2) the inhibition constants of the products, and 3) knowledge of whether or not the reaction is reversible. Therefore, in order to address these issues, product inhibition studies were employed and the ability of the enzyme to catalyze the synthesis of glutamine and LGH was examined.


Materials and Methods

Chemicals and Reagents
[14C]-glutamine was purchased from Amersham. Scintillation fluid, ScintiVersTM II* was obtained from Fischer (Orlando, FL). DE81 anion exchange chromatography paper was purchased from Whatman (Hillsboro, OR). Other chemicals, including L-glutamine and L-glutamic-y-monohydroxamate, and hydroxylamine were obtained from SIGMA Chemical Company (St. Louis, MO) and were of the highest commercial purity.
Enzyme Preparation
Construction of recombinant wild-type AS-B and the mutant AS-B in which R30 has been replaced by alanine (R30A) have been described elsewhere (Boehlein et al., 1994a,b). Both wild-type and mutant proteins were expressed., and purified using standard




47



procedures (Boehlein et al., 1994a). Protein concentrations were determined by the method of Bradford (1976) using an assay kit supplied by Bio-Rad and mouse immunoglobulin G to construct a standard curve. During the course of these investigations an alternate method of determining the protein concentration revealed that the current use of mouse immunoglobulin G as a standard gives erroneously high protein concentrations for AS-B. Amino acid composition analysis of AS-B, carried out by the Protein Chemistry Core Facility of ICBR at the University of Florida, showed that the actual value for the concentration of this enzyme is 2.7 times smaller than that obtained using the Bradford assay. Therefore, all of the values of enzyme concentration and therefore all kcat and kcat/Km values given in this chapter have been corrected by dividing the protein concentration by the factor 2.7. Glutaminase Assays
Initial rates of L-glutamine and L-glutamic--monohydroxamate (LGH) hydrolysis were determined by a modified procedure which uses glutamate dehydrogenase in the presence of NAD+ to measure glutamate concentration (Bernt & Bergmeyer, 1974). Reactions (100 tl) containing 100mM Bis-Tris and Tris-HC1 (pH 8.0) each, 8mM MgC12 and various concentrations of glutamine or LGH were initiated by the addition of wild-type or mutant R30A AS-B and incubated at various temperatures (4C-40C). A control reaction lacking enzyme was used to monitor any glutamate formed from spontaneous breakdown. The reactions were terminated by the addition of 20 tl of IN acetic acid. A coupling reagent (300 mM glycine, 250 mM hydrazine, pH 9, 1 mM ADP, 1.6 mM NAD+ 2.2 units of glutamate dehydrogenase) was added




48


and incubated 10 minutes at room temperature. The absorbance at 340 nm was measured and the concentration of glutamate was determined by comparison to a standard curve. Initial velocities were measured at 9 different substrate concentrations and each velocity was an average of 3 measurements. Stability of the wild-type and mutant AS-B enzymes at each temperature was confirmed by incubation in buffer of the appropriate temperature for various amounts of time followed by an assay of activity at pH 8. Initial velocity conditions were confirmed at each pH by linear plots of v versus time and v versus enzyme concentration. The pH of the buffer was adjusted to pH 8 at each temperature studied to account for the pKa change with temperature.
Determination of I- for glutamate and hydroxylamine in the AS-B catalyzed synthesis of LGH
The Michaelis constant, KM, of glutamate in the synthesis of LGH was determined by incubating wild-type AS-B with various concentrations of glutamate and a saturating concentration of hydroxylamine (150 mM) in Tris-HC1, pH 8.0, at 37'C for 15 min. In a similar manner the Km for hydroxylamine was determined by incubating the wild-type enzyme with various concentrations of hydroxylamine and a saturating concentrations of glutamate (150 mM) in Tris-HC1, pH 8.0, at 37C for 20 min. In both cases, the 300 ptl reaction was terminated by the addition of 100 tl of 16 % TCA. LGH formation was determined in a final volume of 500 tl by adding a solution containing 80% TCA, 6N HC1, and 10% FeC13 in 0.02 N HC1 to the remaining reaction, centrifuging the samples in a microcentrifuge to remove particulates, and measuring the absorbance of the




49



hydroxamate-FeC13 complex at 540 nm. A standard curve using a stock solution of authentic LGH was used to quantitate the product formed in these reactions.
The ability of AS-B to catalyze the synthesis of glutamine also was examined. Wild-type AS-B (37 jig) was combined with glutamate and ammonium chloride (100 mM each) in Tris-HC1 (200 mM) in a total volume of 50 jil. The reactions were incubated for 4 hours at 370C followed by the addition of 20 tl of 2N acetic acid. The terminated reactions were filtered through a 2 jim filter and derivatized with phenylisothiocyanate (PITC). The glutamine and glutamate derivatives were separated by HPLC and quantitated spectroscopically. This assay procedure can detect less than 100 pmoles under our standard procedures Product inhibition of the glutaminase reaction
Wild-type AS-B was incubated in a 100 jtl reaction containing various concentrations of [4C] glutamine (SA 880-20,000 dpm/nmol) in 100mM Bis-Tris and Tris-HC1 at 37C for 10 min in the presence of various concentrations of glutamate or NH4Cl. The reactions were terminated by the addition of 10 jil of 1N acetic acid. Glutamate was separated from glutamine on Whatman DE-81 ion-exchange chromatography paper using deionized distilled water as the mobile phase. After 6 hours, elution was stopped, and the paper was dried and cut into two strips to separate glutamine from glutamate. The radioactivity associated with each strip was counted in ScintiVersll* scintillation fluid on a Beckman LS 61C scintillation counter. Control experiments were carried out in an identical manner except that the enzyme was excluded from the reaction mixture. The separation of




50



the amino acids was visualized in a non-radioactive control by spraying the paper with 0.2% ninhydrin in 95% ethanol. Glutamine was found to migrate with the solvent front while glutamate remained at the origin. The amount of "4C-labeled glutamate formed in the reaction was determined by subtracting the percentage of total radioactive counts located at the origin in the control reactions from the percentage in each sample.
In a similar manner, the inhibition of glutamate also was examined at 5'C except that the reaction time was 40 min. Data Analysis

The values of Kmn and kcat were determined from least-squares fit to vo vs [S] plots using the Prism software package supplied by Graphpad, Inc., (San Diego, CA) according to equation 1: V ma x [S]
v = (1)
Km + [S](

Where v is the initial velocity, [S] is the concentration of substrate, Vmax is the maximal velocity, and Km is the Michaelis constant.
As shown in the equation below, the value for -Ea/RT can be derived from the slope of a plot of the log kcat versus l/T (K) where A is a constant which is related to the collision frequency and steric factors.
k = Ae(-Ea/RT) (2)
Values for the energy, enthalpy, and entropy of activation then were calculated using the following equations:
AH* = Ea RT (3)
AG* = RT ln(k3T/h) RTln(k) (4)




51



AG* = AH* TAS* (5)
where AH* is the enthalpy of activation, R is the gas constant, kB is boltzmans constant, h is Planck's constant, k is the forward rate constant; AG* is the free energy of activation, and AS' is the entropy of activation.


Results
Effect of temperature on glutamine and LGH hydrolysis catalyzed by wild-type AS-B and the AS-B mutant R30A

The steady-state kinetic parameters of glutamine and LGH
hydrolysis were determined at a range of temperatures from 5' C to 400 C. The Arrhenius plots (Figures 3.1 & 3.2) were observed to be linear indicating either that there was no change in the ratedetermining step over the temperature range studied or that the activation energies for the limiting steps were very similar.
The temperature dependence of the catalytic rate constants of the R30A-catalyzed hydrolysis of glutamine also displayed linearity in the Arrhenius plot, as shown in Figure 3.3. The activation parameters are given in Table 3.1. LGH synthesis catalyzed by wild-type AS-B
AS-B was found to catalyze the formation of LGH from
glutamate and hydroxylamine. The reaction rate of 0.29 sec' (Table
3.2, Figures 3.4 & 3.5) was about 12% of that of the corresponding forward rate of 2.4 sec1 at pH 8 (see chapter 2, Table 2.3), and was fast enough to obtain the kinetic parameters of LGH synthesis catalyzed by wild-type AS-B (Table 3.2).
The steady-state kinetic parameters of reversible reactions can




52


be used to calculate the equilibrium constant for the overall reaction using the Haldane relationship for a one-substrate/two-product reaction given in equation (6) (Cleland, 1963):


VfKip Vf Kp Kiq
Keq K -(6)
VrKia Vr Ka



where Keq is the equilibrium constant for the conversion of substrate to product, V, and V, represent the forward and reverse reaction rates, respectively, and the indices p, q, and a refer to hydroxylamine, glutamate and LGH, respectively. Using equation (6) and the following parameters, obtained at 37oC: k'r = 2.4 sec-', Km(NH2OH)= 95 mM, Ki(gu,)=182 mM, k'cat= 0.29 sec1, Ki(LGH)= 0.83 mM; a value of Keq=172 was calculated. Using this value for Kcq and the equation, AGo = -RTlnKeq, the difference in free energy between substrates and products, AGO, was determined to be -13.2 kJ/mol K. Product inhibition glutamine hydrolysis catalyzed by wild-type AS-B
Figures 3.6 and 3.7 show the plots for the inhibition of the
glutaminase reaction by glutamate at 5oC and 37oC, respectively. The Ki values for glutamate determined from the replot of the slope versus glutamate concentration (Figure 3.8 & 3.9) at 50C and 37oC were 260 mM and 182 mrM, respectively. An attempt to obtain the Ki for ammonia was made. However, NH4C1 concentrations up to 200 mM seemed to have little effect on the rate of the glutaminase reaction.




53









2


C 1



0 .j
-1



-2
0.00300 0.00325 0.00350 0.00375
1/'1(K)







Figure 3.1: Temperature dependence of glutamine hydrolysis catalyzed by wild-type AS-B. The initial rates were determined at pH 8.0 in 100 mM Bis-Tris and Tris-HCI as described in the materials and methods.




54















1



0
.i
-1




-2-r
0.0032 0.0033 0.0034 0.0035 0.0036 0.0037
1/T (K)


Figure 3.2: Temperature dependence of LGH hydrolysis catalyzed by wild-type AS-B. The initial rates at each temperature were determined at pH8.0 in 100 mM Bis-Tris and Tris-HC1 as described in materials and methods.




55
















0






-2



0.00300 0.00325 0.00350 0.00375
1/T (K)


Figure 3.3: Temperature dependence of glutamine hydrolysis catalyzed by the AS-B mutant R30A. The initial rates at each temperature were determined at pH 8.0 in 100 mM Bis-Tris and Tris-HCI as described in materials and methods.




56














Table 3.1: Thermodynamic properties of glutamine and LGH hydrolysis catalyzed by wild-type AS-B and the R30A mutant.


Substrate/ AG* AH* AS* TAS'
enzyme kJ mol' kJ mol' J mol-'K-' kJ mol'
(370C) (370C) (5-400C) (370C)
gln/wt 167 89 -252 -78
LGH/wt 152 82 -226 -70
gln/R30A 147 77 -226 -70


The initial rates were determined at pH 8.0 in 100 mM Bis-Tris and Tris-HCI at temperatures ranging from 5-400C, under conditions described in the materials and methods.




57








200



S150
E

S100



50


0
0 50 100 150
[NH2OH] mM








Figure 3.4: Effect of hydroxylamine concentration on the synthesis of LGH catalyzed by wild-type AS-B. Wild-type AS-B (18.5 4g) was incubated with various concentrations of hydroxylamine and saturating concentrations of glutamate (150 mM) in Tris-HC1, pH 8.0 at 370C for 20 min. The reaction was terminated by the addition of 16% TCA and LGH formation was measured as described in materials and methods.




58







250


200

E
150

0
E
S100


50



050 7'5 100 125
(Glu] mM








Figure 3.5: Effect of glutamate concentration on the synthesis of LGH catalyzed by wild-type AS-B. Wild-type AS-B (18.5 gg) was incubated with various concentrations of glutamate and a saturating concentration of hydroxylamine (150 mM) in Tris-HC1, pH 8.0 at 370C for 15 min. The reactions were terminated by the addition of 16% TCA and the formation of LGH was measured as described in materials and methods.





59














Table 3.2: Kinetic constants for the synthesis of LGH catalyzed by wild-type AS-B.


Substrate kcat (sec-') K,1 (rnaM) kcat/K (M-1 sec')

glutamate 0.36 0.02 76 5 4.7
hydroxylamine 0.29 0.01 95 7 2.9


The initial rates were determined at pH 8.0 in 100 mM Bis-Tris and Tris-HC1 at 371C, by varying one substrate while keeping the second substrate saturating. The conditions of the reaction and the determination of the LGH formed in the reaction are described in the materials and methods.




60








0.3


E
~0.2
E 0

%m 0.1Ii I I I i
-5 0 5 10 15 20 25

1/[GIn] mM1





Figure 3.6: Glutamate inhibition of the glutaminase reaction catalyzed by wild-type AS-B at 5C. The rate of glutamine hydrolysis was determined as described in the materials and methods in the presence of 0 (i), 10 (A), 50 (V), 100 (*), and 136 (0) mM glutamate.




61












0.00250.00200.00150.00100.0005

II I I
-1 0 1 2 3

1/Gin (mM-1')


Figure 3.7: Glutamate inhibition of the glutaminase reaction catalyzed by wild-type AS-B at 37oC. The rate of glutamine hydrolysis was determined as described in the materials and methods in the presence of 0 (U), 40 (*), 80 (A), and 100 (V) mM glutamate.




62











0.0150 0.01250.0100O 0.00750.00500.00250.0000 ,
0 25 50 75 100 125 150
[Glu] mM


Figure 3.8: Replot of the slope from the double-reciprocal plots of glutamine hydrolysis at 50C versus glutamate concentration.




63













0.00080.0006,, 0.00040.00020.0000 I I I I I
0 25 50 75 100 125
[Glu] mM


Figure 3.9: Replot of the slope from the double-reciprocal plots of glutamine hydrolysis at 37oC versus glutamate concentration.




64



Discussion

Many of the experiments in the following chapters required a reaction rate slower than that observed at 37'C, the standard temperature at which the reaction is examined. One way to obtain a slower rate is to examine the reaction at low temperatures therefore, many of the experiments in the following chapters have been conducted at 50C. In preparing for these experiments it was important to know whether the results obtained at 5'C could be compared to those at the standard temperature. Temperature variations often cause changes in the rate-limiting step of a reaction. Since non-linear Arrhenitis plots are indicative of such changes, the temperature dependence of the glutaminase reaction was examined to confirm that there was not a change in the rate limiting step between 5'C and 37'C. In the case of the glutaminase reaction, it is difficult to conclude that a single step is rate determining across the entire range of temperatures studied. Certainly, the linear Arrhenius plots suggest that a change in the rate limiting step between reactions at 5'C and 37'C is unlikely. However, it should be noted that two steps in the reaction may have similar activation energies, and therefore a change in rate limiting step would remain unnoticed in these experiments. Such an event is a possibility in AS-B catalyzed glutamine hydrolysis as demonstrated by the unexpected similarity in activation constants for reactions catalyzed by wild-type and the mutant R30A (Table 3.1 and Figures

3.1 and 3.3). Partitioning experiments in chapter 4 imply that in reactions catalyzed by R3OA, hydrolysis of the thioester is rate limiting. In contrast, direct measurements of the deacylation step of glutamnine hydrolysis suggest that an additional step contributes to the




65



overall rate of the reaction catalyzed by the wild-type enzyme. The differences in the nature of the rate determining step in the reactions catalyzed by the wild-type and mutant enzyme were expected to be reflected in the activation parameters. The lack of any differences in the parameters suggests that the two slow steps in the wild-type catalyzed reaction have similar activation energies.

The patterns of product inhibition can provide information on the order of product release. However, the studies of product inhibition presented here were inconclusive due to the inability of ammonia to inhibit the glutaminase reaction. On the other hand, the examination of glutamate inhibition provided a needed inhibition constant which will be used in the model described in chapter 7.

The inability of ammonia to inhibit the glutaminase reaction and the lack of detectable glutamine synthesis using ammonia and glutamate as substrates suggests that the acylation step is essentially irreversible under the conditions of the examination. With the ability to detect glutamine concentrations to 0.1 nmol (see materials and methods), an upper limit on the turnover number of kcat< 5xl104 sec-1 can be set for glutamine synthesis at 37'C catalyzed by the wild-type AS-B under the conditions studied. In contrast, the reversibility of LGH hydrolysis catalyzed by wild-type AS-B was easily demonstrated and has a keat which is 12% of the forward reaction. The difference in the ability to synthesize the reverse reaction may be a reflection of the greater nucleophilicity of hydroxylamine. The kinetic parameters, kcat and Kin, obtained from the analysis of LGH synthesis allowed the determination of the equilibrium constant for the overall reaction.




66


The free energy, AGO, derived from this equilibrium constant was calculated to be -13.2 kJ/mol.












CHAPTER 4
DETERMINATION OF THE RATE DETERMINING STEP IN ASPARAGINE
SYNTHETASE B-CATALYZED GLUTAMINE HYDROLYSIS


Introduction
Asparagine synthetase B (AS-B) is thought to be organized into two functional domains: the glutamine amide transfer (GAT) domain, which catalyzes the hydrolysis of glutamine to glutamate; and the synthetase domain, which catalyzes the formation of a 3-aspartylAMP intermediate and the synthesis of asparagine. While the overall reaction involves three substrates, four products, and possibly twenty rate constants; the ability of the enzyme to catalyze glutamine hydrolysis in the absence of the other substrates provides the opportunity to examine glutamine utilization under a simpler one substrate, two product reaction. A detailed description of this reaction would provide a useful tool in understanding the roles played by conserved amino acid residues of the GAT domain in glutamine utilization. Therefore, this chapter presents a first step towards characterizing the glutaminase reaction catalyzed by AS-B.
Analysis of the amino acid sequence of the GAT domain suggests that AS-B belongs to a family of enzymes known as the glutamine amidotransferases (Zalkin, 1993). Like AS-B, the enzymes of this family catalyze nitrogen transfer using glutamine as the primary source of nitrogen. While all enzymes in the glutamine amidotransferase family contain a cysteine which is essential for

67




68


glutamine dependent activity, the position of this cysteine as well as other sequence differences in the GAT domain further divides the family into two classes. The essential cysteine residue is located within the GAT domain for class I enzymes whereas it resides at the N-terminus in the class II enzymes such as glutamine phosphoribosylpyrophosphate amidotransferase (GPA) (Tso et al., 1982) glucosamine-6-phosphate synthetase (GFAT) (Badet et al., 1987), and asparagine synthetase (Humbert et al., 1980). Initially, site directed mutagenesis studies suggested that the N-terminal cysteine as well as a histidine and aspartate residue were involved in the reaction catalyzed by GPA (Mei & Zalkin, 1989). As this catalytic triad is found in many thiol proteases (Brocklehurst et al., 1987), it was proposed that the glutamine hydrolysis reaction catalyzed by the class II enzymes occurs via a mechanism analogous to that of peptide hydrolysis catalyzed by the thiol proteases, such as papain. This hypothesis was extended to the class I enzymes as well (Mei & Zalkin, 1989). Since then, the importance of a catalytic triad composed of a conserved cysteine, histidine, and glutamate residues has been demonstrated for the class I enzymes through site-directed mutagenesis (Amuro et al., 1985; Miran et al., 1991) and x-ray crystallography (Tesmer et al., 1996; Thoden et al., 1997).
On the other hand, the picture remains more ambiguous for the class II amidotransferases as subsequent studies confirmed only the importance of the conserved N-terminal cysteine residue (Boehlein et al., 1994a). In fact, a more recent alignment of the GAT domain which includes a greater number of amidotransferase sequences reveals that there are no histidines which are conserved across this




69


entire class of enzymes (Boehlein et al., 1994a; Zalkin, 1993). Moreover, studies using site-directed mutagenesis of AS-B (Chapter 2; Boehlein et al., 1994a) and x-ray crystallography of GFAT (Isupov et al., 1996) and GPA (Kim et al., 1995) suggest that the class II amidotransferases function differently than papain with respect to acid-base catalysis. While the lack of a conserved histidine in the class II amidotransferases contrasts with the essentiality of such a residue in thiol proteases, recent evidence for an acyl enzyme intermediate (chapter 5 & 6) formed during glutamine hydrolysis catalyzed by AS-B provides a basis for comparison of the hydrolysis reactions catalyzed by the two enzyme families. A minimal description of amide hydrolysis catalyzed by papain, which occurs via a thioester intermediate (E-TE), is shown in equation 1 below:


k, k
S + ES --2-- E-TE E+P2 (equation 1)
k -1 + P t

In relating this equation to glutamine hydrolysis catalyzed by AS-B, ES is the enzyme-substrate complex, E-TE is the acylenzyme intermediate, and P1 and P2 are ammonia and glutamate, respectively. In an effort to understand the mechanism of glutamine hydrolysis catalyzed by AS-B, the glutaminase reaction will be examined in order to develop a model for this particular aspect of the enzymatic reaction. The minimal model shown above will be used as a starting point in this investigation to provide evidence (in addition to that shown in chapter 5 for the existence of a thioester intermediate, to determine whether steps leading to its formation or




70


those involved in its breakdown are rate limiting, and to compare and contrast catalytic behavior of three groups of hydrolytic enzymes: a class II amidotransferase (AS-B), a class I amidotransferase (CAD), and a thiol protease (papain).


Materials and Methods
Chemicals and Reagents
14C-glutamine was purchased from Amersham. Scintillation fluid, ScintiVersTM II* was obtained from Fischer (Orlando, FL). DE81 anion exchange chromatography paper was purchased from Whatman (Hillsboro, OR). Other chemicals, including L-glutamine and L-glutamic-y-monohydroxamate, hydroxylamine, and 6-diazo-5oxonorleucine (DON) were obtained from SIGMA Chemical Company (St. Louis, MO) and were of the highest commercial purity. Enzymes
The construction of recombinant AS-B and the AS-B mutant,
R30A, has been described previously (Boehlein et al., 1994a, 1994b). The enzymes were obtained by overexpression and purified using standard procedures (Boehlein et al., 1994a). Protein concentration was determined by the method of Bradford (1976) using an assay kit supplied by Biorad and immunoglobulin G as a standard. Recent examination of the protein concentration using amino acid analysis of AS-B, performed by the Protein Core Facility of ICBR at the University of Florida, gave a value for protein concentration which was 2.7-fold lower than that obtained with the previous method. Therefore, in this report the enzyme concentration has been corrected by dividing the enzyme concentrations by the factor 2.7.




71


Steady State Kinetics
The kinetic parameters of AS-B-catalyzed glutamine, LGH, and L-glutamine-hydrazide hydrolysis were obtained in an end-point assay which uses glutamate dehydrogenase in the presence of NAD' to measure the concentration of glutamate formed (Bernt & Bergmeyer, 1974). Reactions (100 pl) containing 100mM Bis-Tris and Tris-HCI (pH 7.5) each, 8mM MgCl2 and various concentrations of glutamine, LGH, or glutamic acid-y-hydrazide were initiated by the addition of 1.5 gg wild-type AS-B and incubated at 370C. After 10 minutes, the reactions were terminated by boiling for 2 minutes. Reactions in which the enzyme was added to the mix during the termination period of boiling were used to monitor any glutamate formed from spontaneous breakdown. A coupling reagent (300 mM glycine, 250 mM hydrazine, pH 9, 1 mM ADP, 1.6 mM NAD+ 2.2 units of glutamate dehydrogenase) was added to the reaction and the resulting mixture was incubated 10 minutes. The absorbance at 340 nm was measured and the concentration of glutamate was determined by comparison to a standard curve. Initial velocities were measured at 9 different substrate concentrations and each velocity was an average of 2-4 measurements.
The values of Km and kcat were determined from least-squares fit to vo vs [S] plots using the Prism software package supplied by Graphpad, Inc., (San Diego, CA).
Determination of the K. for glutamine and LGH
The rate of inactivation by the glutamine analog, 6-diazo-5oxonorleucine (DON), was determined by incubating 30 gtg wt AS-B in a 300 p1 solution containing 100 mM Bis-Tris and Tris-HC1, pH 8 and




72


varying concentrations of DON at 5C. At various time points 20 ptl aliquots were removed from the incubation mixture and the residual activity was measured in 200 pi buffer containing 12 mM glutamine. Control experiments measuring the activity in the presence of diluted inhibitor concentrations verified that DON did not interfere in the assay of residual activity.
Glutamine protection of DON inhibition was examined as
described above except that wt AS-B was incubated with varying concentrations of DON in the presence of either 20 ptM or 50 tM glutamine. Alternatively, LGH protection of DON inactivation of the wild type enzyme and glutamine protection of R30A inactivation were examined by incubating wild-type or R30A AS-B with a constant concentration of DON (6 4M for wild type and 650 tM for R30A) and varying concentrations of LGH or glutamine, respectively. An analysis of glutamate formation in AS-B catalyzed glutamine hydrolysis as a function of time.
Glutamine hydrolysis catalyzed by wt AS-B was examined at 50C, a temperature at which the reactions were slow enough to mix by hand. The reactions were initiated by adding 0.37 or 0.74 nmol wt AS-B to a 50 pL1 solution containing 100 mM Bis-Tris and Tris-HCl (pH 8) and ['4C]-glutamine (2mM, SA = 9 nCi/tl). After the appropriate amount of time, the reactions were terminated with 20 pI of 2N acetic acid. Glutamate was separated from glutamine on Whatman DE-81 ion-exchange chromatography paper using deionized distilled water as the mobile phase. After 6 hours, elution was stopped, and the paper was dried and cut into two strips to separate glutamine from glutamate. The radioactivity associated




73


with each strip was counted in ScintiVerslI* scintillation fluid on a Beckman LS 61C scintillation counter. Control experiments were carried out in an identical manner except that the enzyme was excluded from the reaction mixture. The separation of the amino acids was visualized in a non-radioactive control by spraying the paper with 0.2% ninhydrin in 95% ethanol. Glutamine was found to migrate with the solvent front while glutamate remained at the origin. The number of nanomoles of 4C-labeled glutamine in each sample was determined by subtracting the percentage of total radioactive counts located at the origin in the control reactions from the percentage in each sample. All values are derived from the average of four separate reactions.
To examine the effect of glutamate on the burst, wild-type AS-B was incubated on ice either alone or with 100 mM L-glutamate, or DGlutamate for 3 hours prior to the reaction. The enzyme/glutamate mixture then was added to the reaction and product formation was determined at various time points as described above. Hydroxylamine Partitioning of the Thioester

A reaction mixture containing either wt AS-B (9.3 kg), or R30A (18.5 gg), and 50 mM glutamine, 100 mM Bis-Tris and Tris-HC1 (pH 8) and a variable concentration of hydroxylamine in a total volume of 300 p.l was incubated at 37C for 45 minutes. The reaction was terminated by the addition of 100 p.l of 16% TCA and a 50 p.l aliquot was removed to determine the amount of glutamate formed using the previously mentioned GDH assay. LGH formation was determined in a final volume of 500 p.1 by adding a solution containing 80% TCA, 6N HCI, and 10% FeCI3 in 0.02 N HC1 to the remaining reaction,




74


and measuring the absorbance of the hydroxamate-FeC13 complex at 540 nm. A standard curve using a stock solution of authentic LGH was used to quantitate the product formed in these reactions.


Results
Partitioning of a thioester intermediate to 7-glutamvl hydroxamate

The minimal mechanism, given in equation 1, requires the formation of a covalent intermediate. Due to the highly reactive nature of hydroxylamine towards acyl groups, this agent has been used to provide evidence for an acylenzyme intermediate and to determine whether its formation or breakdown is rate limiting in hydrolysis reactions catalyzed by serine proteases (Caplow et al., 1964; Epand et al., 1963) as well as by the amidotransferase CAD (Chaparian et al., 1991). Since hydroxylamine competes with H,O in the deacylation step, its presence will cause an increase in the amount of total product formed (hydroxamate plus glutamate) only if this same step is rate limiting (reaction 2). In contrast, if the acylation step is very slow then the presence of hydroxylamine will not have an effect on the amount of total product produced as the slow rate of the acylation step will limit the amount of thioester available for hydrolysis and hydroxaminolysis.
H20 E + Glu
K, k2
E +Gln E.Gln E-TE (Reaction 2)

NH2OH E + LGH




75


In an attempt to show that AS-B catalyzed hydrolysis occurs via a thioester intermediate and to determine which step in hydrolysis is rate limiting, the effect of various concentrations of hydroxylamine on the glutaminase reaction was examined. As shown in Figure 4.1, even very low concentrations of hydroxylamine were sufficient to partition the reaction towards the production of glutamate hydroxamate. Unexpectedly however, an increase in hydroxylamine concentration in the AS-B catalyzed glutaminase reaction caused a decrease in total product formed (figure 4.1).

One potential cause for this decrease in total product formed in the presence of hydroxylamine is a slow release of L-glutamate hydroxamate due to its tight binding to the enzyme. To test this idea, the partitioning experiment was repeated using an AS-B mutant (R30A) which exhibits poor substrate binding. A role in substrate binding has been implied for Arginine-30 in AS-B (Boehlein et al., 1994b) and the cognate arginine in GFAT (Isupov et al., 1996) in studies using site-directed mutagenesis and x-ray crystallography, respectively. Use of R30A in the partitioning experiments resulted in an increase in total product with increasing concentrations of hydroxylamine (figure 4.2).
Comparison of AS-B catalyzed hydrolysis of three substrates
The kinetic constants kcat, Km, and kcat/Km for the AS-B catalyzed hydrolysis of L-glutamine, L-glutamic acid-y-monohydroxamate, and L-glutamic acid-y-hydrazide are given in Table 4.1. As shown in the table, the kcat values for the three substrates were similar while each displayed different kcat/Km values.




76






600
i.

500

) 400E

5g 300. 200100


0 10 20 30 40 50 60 70
[NH2OH] (mM)




Figure 4.1: Partitioning of the thioester intermediate to L-glutamicy-monohydroxamate using hydroxylamine in a reaction catalyzed by wt AS-B. A mixture containing wt AS-B, 50 mM glutamine, 100 mM Bis-Tris and Tris-HCI (pH 8), and the indicated concentrations of hydroxylamine was incubated at 370C for 45 min. The reactions were terminated by the addition of 4% TCA. An aliquot was removed to determine glutamate formed (0) by an end-point assay using glutamate dehydrogenase. LGH formation (A) was measured by adding a FeCl2 solution and measuring the absorbance at 540 nm as described in materials and methods. The nmol of LGH and glutamine formed at each concentration of hydroxylamine were added to give the total product formed (U).




77








2000


1500
E

4-E 10000
L
500


0
0 10 20 30 40
[NH2OH] mM




Figure 4.2: Partitioning of the thioester to glutamate hydroxamate using hydroxylamine in the glutaminase reaction catalyzed by AS-B R30A. A mixture containing wt AS-B, 80 mM glutamine, 100 mM Bis-Tris and Tris-HCI (pH 8), and the indicated concentrations of hydroxylamine was incubated at 370C for 45 min. The reactions were terminated by the addition of 4% TCA. An aliquot was removed to determine glutamate formed (A) by an end-point assay using glutamate dehydrogenase. LGH formation (0) was measured by adding a FeC1, solution and measuring the absorbance at 540 nm as described in materials and methods. The nmol of LGH and glutamine formed at each concentration of hydroxylamine were added to give the total product formed (+).




78













TABLE 4.1:
Kinetic constants for wt AS-B-catalyzed hydrolysis of L-glutamnine, Lglutamic acid-y-monohydroxamate, and L- glutamic acid-,y- hydrazide.




Substrate k~(e) K (M) ki. K sM se c'

L-glutamine 1.9 0.03 1.0 0.06 1900

L-glutamic-y-monohydroxamate 2.5 0.02 0.65 0.02 3846

L-glutamic-y-hydrazide 2.0 0.05 2.3 0.20 869




79


Determination of the Dissociation constants for glutamine and LGH using the covalent modifier. 6-diazo-5-oxonorleucine (DON).
The diazo ketone analogue of glutamine, DON, has been shown to inhibit the glutaminase activity of bovine AS in such a manner that the effects could be eliminated only by the addition of glutamine and not by desalting (Mehlhaff & Schuster, 1991). Furthermore, the x-ray structure of the glutamine active site labeled by DON in another class II amidotransferase, E. coli GPA (Kim et al., 1996), has provided evidence that this analog alkylates the cys-1 residue in a reaction which is comparable to the formation of the glutamylenzyme intermediate. Using the method of Meloche (1967), the affinity analogue, DON, has been used in the determination of the glutamine dissociation constant for the glutaminase reaction catalyzed by anthranilate synthetase (Goto et al., 1976), CAD glutaminase (Chaparian & Evans, 1991) and the amidotransferase PabA/PabB complex (Roux & Walsh, 1992). Likewise, the ability of DON to inactivate wild-type and the R30A mutant of AS-B was examined and the effect of glutamine or LGH on the inactivation was used to determine the dissociation constant of glutamine and LGH. Incubation of wild type AS-B with DON prior to the measurement of the glutaminase activity caused a time-dependent loss of activity over a period of 20 minutes. The semilogrithmic plot of the residual activity versus time shows that DON inactivates AS-B with pseudofirst order kinetics (figure 4.3). This plot also shows that inactivation is irreversible since the residual activity of the enzyme after it had been incubated with DON was determined using 20 mM glutamine, providing a 1400-fold dilution. The inactivation half-times, u, were




80


determined from these plots and were used in a replot to determine the Ki for DON according to the following equation described by Meloche (1967):


(TKi)+ (1)



where u is the time required for the inhibitor to cause a 50% loss of activity (tr= n2/first order rate constant for inactivation); [I] is the inhibitor concentration; T is the time required for one-half inhibition at infinite inhibitor concentration; and Ki is the inhibition constant for the inhibitor. When measuring the residual AS-B glutaminase activity after incubating the enzyme with DON, the replot (tc vs 1/[I]) showed a straight line which extrapolated to a minimum inactivation half-time, T, of 1.22 minutes (figure 4.4). The Ki for DON determined from the slope of this replot was 34 .M.
The dissociation constant for glutamine, K was determined by measuring the competitive binding of glutamine and DON. Therefore, DON inactivation was examined in the presence of 20 jiM and 50 jM glutamine (Figures 4.5 and 4.6). Using the following equation (Meloche, 1967):


TK( 1+ IS) (2)
=[I]




81









2.25
[DON]
>12.00 1 4 iM
A~ 6pM
1.75
03 8 PvM

0 1.50- 0 10OPM
0)
1.25-012p
0 1 4 pM

1.00
0 5 10 15 20 25

time (min)






Figure 4.3: DON inactivation of AS-B in the absence of glutamine. Wild type AS-B was incubated with the concentrations of DON given in the figure. At time points ranging from 0-20 minutes, an aliquot was removed from the mixture and tested for residual activity in a reaction containing 12 mM glutamine. The glutaminase activities of the DON modified enzymes were measured as described in materials and methods.




82












2015


10


5


0
0.00 0.05 0.10 0.15 0.20 0.25
1/DON RM





Figure 4.4: Replot of DON inactivation of AS-B in the absence (0) and presence of glutamine (A. 20 pM; 0, 50 tM).




83


a replot of 'r vs 1/[I] at each concentration of glutamine gave straight lines with a different slope for each glutamine concentration (Figure

4.4). The slope of the line resulting from 20 RLM glutamine is 50.73 and that from 50 LM glutamine is 65.02 corresponding to K, values of 140 iM and 200 jiM respectively.
In a similar manner, DON inactivation and LGH protection against inactivation was examined for wild type AS-B, and DON inactivation and glutamine protection against inactivation was examined for the AS-B mutant R30A (Figures 4.7-4.14). Using equation (1) and the replot of vs 1/[DON] the minimum inactivation half-time, T, for DON inactivation of wild type catalyzed LGH hydrolysis (Figure 4.8) and R30A catalyzed glutamine hydrolysis (Figure 4.12) were 1.4 sec and 3.5 sec, respectively. The K for DON in these two reactions, determined from the slope of the replot was 28 M for LGH hydrolysis and 8.7 mM for inactivation of R30A catalyzed glutamine hydrolysis. The following equation was used to determine the dissociation constant for LGH in the wild type catalyzed glutaminase reaction and for glutamine in the R30A catalyzed reaction:


c=[S]T](Kac) + (T+T Kinact) (3)
KSM



The Ks, derived from the slope of the line in a replot of c vs [substrate] for LGH in the wild type catalyzed hydrolysis reaction (Figure 4.10) was 70 jtM and for glutamine in the R30A catalyzed reaction (Figure 4.14) was 9.5 mM




84










2.25- [DON]

2.00 6 gM
.8gM
1.75 10 gM

Co
0
1.25

1.00
0 5 10 15 20 25

Time (min)




Figure 4.5: DON inactivation of AS-B in the presence of 20 tM glutamine. Wild type AS-B was incubated with the concentrations of DON given in the figure. At time points ranging from 0-20 min, an aliquot was removed from the mixture and tested for residual activity in a reaction containing 12 mM glutamine. The glutaminase activities of the DON modified enzymes were measured as described in materials and methods.




85









[DON]
2.5
4 6pM

A 8 pm
2.0 10pM

V 12pM

0
1.5



60 10 15 20 25

Time (min)





Figure 4.6: DON inactivation of AS-B in the presence of 50 jiM glutamine. Wild type AS-B was incubated with the concentrations of DON given in the figure. At time points ranging from 0-20 min, an aliquot was removed from the mixture and tested for residual activity in a reaction containing 12 mM glutamine. The glutaminase activities of the DON modified enzymes were measured as described in materials and methods.




86










[DON]
2.25

2.00-, 6 M
A 8 M

1.75- 10 RM

-0 1.500
1.25

1.00 ,,,
10 15 20
Time (min)




Figure 4.7: DON inactivation of wild-type catalyzed LGH hydrolysis in the absence of LGH. Wild type AS-B was incubated with the concentrations of DON indicated in the figure. At time points ranging from 0-18 min an aliquot was removed from the mixture and tested for residual activity in a reaction containing 12 mM glutamine. The glutaminase activities of the DON modified enzymes were measured as described in materials and methods.