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Attempt to Crystallize Human Asparagine Synthetase Bound to Two Inhibitors

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Title:
Attempt to Crystallize Human Asparagine Synthetase Bound to Two Inhibitors
Series Title:
Journal of Undergraduate Research
Creator:
Berry, Alexandria H.
Li, Kai
Kursula, Inari
Lindqvist, Ylva
Richards, Nigel G. J. ( Mentor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Language:
English

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serial ( sobekcm )

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Abstract:
Drug resistance in acute lymphoblasitic leukemia (ALL) is an ongoing problem. Resistance to asparaginase (ASNase) treatment – a treatment believed to work by breaking down asparagine in the blood and therefore starving leukemia cells – is linked to up-regulation of human asparagine synthetase (hASNS), the enzyme that produces asparagine. The purpose of this work was to obtain a crystal structure of hASNS with inhibitors bound to both of its active sites. A structure would be used to design potent inhibitors of hASNS to treat ASNase resistance. Conditions for inhibition of the glutaminase active site by 6-diazo-5-oxo-L-norluecine and the synthetase site by a transition state analog, a sulfoximine, were determined. The Ki and and Ki* describing sulfoximine inhibition of DON-inhibited hASNS were found to be 679 nM and 2.9 nM, respectively. The DON-inhibited enzyme was more difficult to inhibit with the sulfoximine than the free enzyme. Milligram amounts of the doubly-inhibited enzyme were screened for crystallization conditions using the sitting drop method. Crystals were obtained, but no structure could be determined. The inhibition studies suggest a possible conformational change in the synthetase site upon the binding of glutamine to the glutaminase site. The crystallization screens revealed increased hASNS solubility at pH 9. From this information, we hope that a crystal structure of hASNS will soon be realized.

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Attempt to Crystallize Human Asparagine Synthetase Bound to
Two Inhibitors





Alexandria H. Berry et al.*



Drug resistance in acute lymphoblasitic leukemia (ALL) is an ongoing problem. Resistance to asparaginase (ASNase) treatment - a
treatment believed to work by breaking down asparagine in the blood and therefore starving leukemia cells - is linked to up-regulation
of human asparagine synthetase (hASNS), the enzyme that produces asparagine. The purpose of this work was to obtain a crystal
structure of hASNS with inhibitors bound to both of its active sites. A structure would be used to design potent inhibitors of hASNS
to treat ASNase resistance. Conditions for inhibition of the glutaminase active site by 6-diazo-5-oxo-L-norluecine and the synthetase
site by a transition state analog, a sulfoximine, were determined. The K, and and K,* describing sulfoximine inhibition of DON-
inhibited hASNS were found to be 679 nM and 2.9 nM, respectively. The DON-inhibited enzyme was more difficult to inhibit with
the sulfoximine than the free enzyme. Milligram amounts of the doubly-inhibited enzyme were screened for crystallization conditions
using the sitting drop method. Crystals were obtained, but no structure could be determined. The inhibition studies suggest a possible
conformational change in the synthetase site upon the binding of glutamine to the glutaminase site. The crystallization screens
revealed increased hASNS solubility at pH 9. From this information, we hope that a crystal structure of hASNS will soon be realized.


Introduction

Human asparagine synthetase and leukemia
In 1970, Cooney and Handschumacher showed
that leukemia cells that were less able to produce
asparagine were more susceptible to chemotherapy [1].
Malignant lymphocytes have also been shown to produce
less asparagine than healthy cells, and therefore must take
asparagine from the blood [2]. L-asparaginase (ASNase) is
an enzyme used to treat acute lymphoblastic leukemia
(ALL) and some forms of acute myeloblastic leukemia
(AML) [3-7]. ASNase treatment is believed to work by
breaking down asparagine in the blood, effectively starving
the leukemia cells [1]. However, 10-12% of patients who
undergo remission after this treatment relapse with
leukemia resistant to ASNase chemotherapy [6, 8-10].


Alexandria H. Berry�, Kai Li�, Inari KursulaL, Ylva
Lindqvist , Nigel G. J. Richards

Department of Chemistry, University of Florida,
Gainesville, FL 32611-7200, USA
�Molecular Structural Biology, Medical Biochemistry and
Biophysics, Karolinska Institutet, S-17177 Stockholm,
Sweden


Human asparagine synthetase (hASNS) produces
asparagine by breaking down glutamine to release
glutamate and ammonia in the N-terminal glutaminase site.
The ammonia travels down a 20 A tunnel to a second
active site, the synthetase site, at the C-terminus. Here,
asparagine reacts with ATP to form an intermediate, P-
aspartal-AMP (PAspAMP), and pyrophosphate. Then,
ammonia attacks pAspAMP to release AMP and form
asparagine [11].
Evidence suggests that up-regulation of human
asparagine synthetase expression may cause ASNase
resistance [12]. Therefore, small molecule inhibitors of
hASNS may be useful as drugs to alleviate this resistance.
In order to design such drugs, detailed knowledge about the
structure of hASNS is necessary. However, over the last
10 years, attempts to crystallize and solve the crystal
structure of the free enzyme have failed.

Inhibition and crystallization of human asparagine
synthetase
The structure of human asparagine synthetase may
facilitate the design of chemotherapeutic agents that may
overcome ASNase resistance. This paper describes the
current attempt to crystallize this enzyme while it is bound
to inhibitors at both active sites. By inhibiting both the
glutaminase and synthetase sites, we hope to lock the
enzyme in its active conformation, facilitating


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
1




ALEXANDRIA H. BERRY ET AL.


crystallization. 6-diazo-5-oxo-L-norleucine (DON), a
glutamine analog, is used to covalently modify the N-
terminal active site. An adenylated sulfoximine, a
transition state analog for the attack of ammonia on
(3AspAMP, is a slow-onset, tight binding inhibitor (Scheme
1), and is used to inhibit the C-terminal synthetase site
[13].


ki [ATP]


E E.A
k2

k4 k3

E.I - El*
k6


k7
kTP E


Scheme 1. Diagram of the kinetic mechanism of the slow-onset, tight
binding inhibition of hASNS by sulfoximine [13, 14].


K (E)(I)
Ki) + (E.I*)
(E.I) + (E.I*)


Kik6

k5 + k6


K, and KI, the initial dissociation and overall
dissociation constant (Equations 1 and 2) for a slow-onset,
tight binding inhibitor, were determined for hASNS
inhibition by sulfoximine according to previously
described methods [14]. Equation 3 can be used to fit the
data obtained by graphing the amount of pyrophosphate
formed during the synthetase reaction versus time while the
enzyme was incubated with various concentrations of the
sulfoximine.


-kt (V - )
[PP] = vst + (1 - e k) (v -
k

(3)

From these graphs, a value for the parameter, k, can be
determined, as can estimates of the initial (vo) and steady-
state velocity (vss) for each reaction. These values are then
used in Equation 4 to determine a value for k6 for each
reaction.


Vss
k6 =k vs
Vo

(4)

The average k6 value is used in Equation 5 to determine k5
and K,. This equation is fit to the data obtained by
graphing the K value determined from Equation 3 versus
the corresponding inhibitor concentration [I]. Here, Ka
represents the Michaelis-Menten constant (0.2 mM in
hASNS) for ATP, and [ATP] represents its concentration;
ATP is a substrate which competes for the same binding
site as the inhibitor (5 mM ATP in hASNS). K,* can then
be determined using Equation 2.


k = k6+ k5 (fP 1

Kai~~I~K Kj)


The kinetic characterization of the inhibition by
sulfoximine will be discussed for two forms of hASNS: (1)
DON-inhibited hASNS, ankd (2) free hASNS. Finally, this
paper will discuss the results of crystallization screens. It
will also describe current work to improve enzyme
solubility. Using the information learned in these
experiments, we hope that a crystal structure of hASNS
will soon be realized.

Materials and Methods

Materials
The adenylated sulfoximine inhibitor was provided
by collaborators (Jun Hiratake, Institute for Chemical
Research, Kyoto University, Japan) as a 1:1 mixture of
diasterioisomers [15]. All other chemicals, including DON
(6-diazo-5-oxo-L-norleucine), were purchased from
Sigma-Aldrich (St. Louis, MO). L-glutamine was
recrystallized as described [16] before its use in all assays.
C-terminally tagged, recombinant hASNS was expressed
using a baculovirus expression system in Sf9 cells as
previously described [17].

Assays
Protein concentrations were determined using a
standard curve constructed with known amounts of bovine
serum albumin [18] using a Bradford assay (Pierce,
Rockford, IL).
The steady-state synthetase activity of hASNS-
which produces asparagine and pyrophosphate at a 1:1
ratio-was determined by continuously monitoring the


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 | Spring 2009
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ATTEMPT TO CRYSTALLIZE HUMAN ASPARAGINE SYNTHETASE BOUND TO Two INHIBITORS


production of inorganic pyrophosphate by measuring the
consumption of NADH at 340 nm (Sigma Technical
Bulletin BI-100). hASNS was added to reaction mixtures
containing 100 mM EPPS, pH 8, 10 mM MgCl2, 20 mM
aspartate, 2.5 mM ATP, and either 100 mM NH4Cl or 20
mM glutamine. All reactions were incubated at 370C and
monitored for 20 minutes. This assay is referred to as the
pyrophosphate assay.
The glutaminase activity of hASNS was assayed
by determining L-glutamate formation using the coupling
enzyme, L-glutamate dehydrogenase (L-GLDH). Reaction
mixtures contained 100 mM EPPS, pH 8, 100 mM NaC1,
10 mM MgC12, and 25 mM L-glutamine to a final volume
of 200 gL. The reactions were initiated upon the addition
of hASNS (1 mg) and incubated at 370C for 10 minutes.
The reactions were quenched by the addition of
trichloroacetic acid to a final concentration of 2.6% (v/v).
The quenched reactions were then added to a second
coupling reaction mixture (300 mM glycine, 250 mM
hydrazine, pH 9, 1.5 mM NAD�, 1 mM ADP, and 2.2 units
of L-GLDH), to a final volume of 1 mL. The absorbance
of each reaction was measured at 340 nm at times 0 and 30
minutes. This process was repeated for the construction of
a standard curve, except that L-glutamine was replaced by
known amounts of L-glutamate.

Activity loss due to incubation
hASNS's loss of glutaminase activity due to
incubation time and temperature was determined by adding
10 gL of deionized water (instead of 10 gL of the inhibitor
solution) to 100 gL of the stock enzyme solution (1 mg/mL
hASNS, 50 mM EPPS, pH 8, 20% glycol (v/v), and 5 mM
DTT). Three aliquots of this solution were then incubated
at 40C, 220C, and 37�C over a period of 61 minutes.
Samples (2 gL) from each aliquot were removed
throughout the 61 minutes, and assayed using the
glutaminase assay as described above.
The loss of ammonia-dependent synthetase activity
due to length and temperature of incubation was
determined by removing 6.5 gL aliquots of stock enzyme
from a -800C freezer at various time points and incubated
at 220C. After all aliquots had incubated for the
predetermined time (0, 10, 20, 30, 40, 50, 60, 65, and 70
minutes), 6 gL of each aliquot was assayed using an
ammonia-dependent pyrophosphate assay as described
above. This same procedure was repeated at 370C over a
60 minute period of incubation (0.0, 2.0, 5.0, 9.9, 14.9,
20.0, 25.0, 30.0, 40.0, and 60.0 minutes).

Inhibition by DON
DON's inhibition of hASNS was characterized by
incubating 45 gL of stock enzyme solution (1 mg/mL
hASNS, 50 mM EPPS, pH 8, 20% glycol (v/v), and 5 mM
DTT), with 5.88 mM DON (final volume of 5 1 gL). A
control incubation solution was set up the same way,
except 100 mM filtered EPPS, pH 8, was used instead of


DON. This filtered EPPS solution was prepared using
Fisherbrand� 25 mm Syringe Filter, 0.2 gm, nylon.
Enzyme solutions were incubated at 220C, and aliquots (15
gL) were removed from the inhibition solution and the
control solution at the same time points (4.2, 6.1, 10.5,
17.8, 22.1, 31.4 minutes). DON was separated from these
aliquots by filtration using 30,000 nominal molecular
weight limit (Daltons) Microcon� Centrifugal Filter
Devices spin columns (Millipore Corporation, Bedford,
MA). Incubation and control aliquots were spun down for
1 minute at 8,000 x g. Afterwards, aliquots were washed
twice by adding 100 gL of 100 mM filtered EPPS, pH 8,
and by spinning down for 4 minutes at 14,000 x g.
Enzyme was recovered by inverting the spin column in a
new microcentrifuge tube, adding 30 gL of 100 mM
filtered EPPS to the bottom side of the filter, and spinning
down for 1 minute at 4,000 x g. All samples were put on
ice and immediately assayed using the glutaminase assay
as described above (10 gL of recovered enzyme in 190 gL
of the initial reaction mixture). The synthetase activity for
the control and inhibited enzyme aliquots was checked
using the pyrophosphate assay, and the Bradford assay was
used to determine the enzyme concentration for all
solutions.

Inhibition of the DON-inhibited enzyme by sulfoximine
The DON-inhibited enzyme was prepared by
incubating stock enzyme with 5.88 mM DON for 20
minutes at 220C. A control incubation solution was also
prepared, and excess DON was filtered off as described
above.
The ammonia-dependent activity of the DON-
inhibited enzyme during incubation with the sulfoximine
inhibitor (stock solution of inhibitor at 0.1 mg/mL in
deionized water) was assayed using the pyrophosphate
assay (incubated at 370C in 100 mM EPPS, pH 8,
containing 10 mM MgCl2, 100 mM NH4C1, 10 mM
aspartate, and 5 mM ATP over a period of 30 minutes with
a 1 mL final volume). The uninhibited control enzyme was
assayed at 0 and 10 mM sulfoximine, while the DON-
inhibited enzyme was assayed at 0, 10, 12,14, 16, and 18
gM sulfoximine. The inhibition of hASNS by the
sulfoximine was characterized as previously described [13,
14].
The glutamine-dependent synthetase activity of the
DON-inhibited enzyme while being incubated with
sulfoximine was assayed (incubated at 370C in 100 mM
EPPS, pH 8, containing 10 mM MgCl2, 20 mM glutamine,
10 mM aspartate, and 5 mM ATP over a period of 20
minutes with a 1 mL final volume). The DON-inhibited
enzyme was assayed with 10 gM sulfoximine, while the
control enzyme was incubated with 0 and 10 gM
sulfoximine.
The glutaminase activity of the dually-inhibited
enzyme was checked using the glutaminase assay as
described above. The DON-inhibited enzyme was assayed


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
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ALEXANDRIA H. BERRY ET AL.


with 0 and 10 gM sulfoximine, while the control, free
enzyme was assayed with 10 jgM sulfoximine.

Large-scale inhibition
Milligram amounts of the DON-inhibited enzyme
were prepared by incubating the stock enzyme solutions
(50 mM EPPS, pH 8, 20% glycol (v/v), and 5 mM DTT)
with 6 mM DON for 30 minutes in 1 mL volumes. Excess
DON was removed by dialysis into 2 L of 50 mM EPPS,
pH 8, and 5 mM DTT for 16 hours at 40C. Dialyzed
enzyme was concentrated using an ammonium sulfate
precipitation (70% saturation of ammonium sulfate) and
resuspended in 50 mM EPPS, pH 8, with 5 mM DTT.
Samples were inhibited with the sulfoximine immediately
prior to crystallization screens by incubation with 20 agM
sulfoximine.

Crystallization screens
Crystallization screens were performed using the
Phoenix Liquid Handling System (Art Robbins
Instruments) using the sitting drop method. Screens from
Qiagen were used, such as the JCSG+ Suite, the pH Clear
Suite, the PEGs Suite, and the Classics Sweet. All Screens
were done in duplicate, one stored at 40C and the other at
250C.


Results

Loss of glutaminase and synthetase activity due to
incubation
hASNS loses activity from incubation at various
temperatures alone, without the addition of inhibitor. In


1
S0.9
t 0.8
* 0.7
co
.E 0.6
E
S 0.5
C 0.4
o 0.3
. 0.2
5 0.1
U_


order to design inhibition experiments where most of the
activity loss was due to inhibition, the enzyme was
incubated at various temperatures as described above
without inhibitor to quantify this activity loss.
Fig. 1 shows the glutaminase activity retained by
hASNS after incubation at 40C, 220C, and 370C. After one
hour at 40C, hASNS still retained 84% of its activity, while
at 370C it retained only 13%. At 220C, however, the
enzyme's glutaminase activity fell to only 57% after one
hour, and was still at 81% after 30 minutes. For this
reason, the glutaminase inhibition by DON was studied at
220C for incubation periods no longer than 30 minutes.
The loss of synthetase activity during various
incubation conditions without the presence of inhibitor
(Fig. 2) showed that the synthetase activity was less fragile
than the glutaminase activity. After 30 minutes at 220C,
the enzyme retained 99% of its synthetase activity, and
89% after 60 minutes. This was important because we did
not want the incubation at 220C for the DON inhibition to
affect the ability of the synthetase site to react. Again,
greater activity loss was shown at 370C; the enzyme
retained 33% of its synthetase activity after incubation at
this temperature for 60 minutes.

Glutaminase inhibition by DON
After 20 minutes at 220C in the presence of 5.88
mM DON, hASNS's glutaminase activity decreased to
10% of the activity of the enzyme under the same
conditions except without DON (Fig. 3). After 20 minutes,
the activity loss slowed and stayed at approximately 10%.
Twenty minutes at 220C was picked as the inhibition
conditions used to prepared the DON-inhibited enzyme for
all further experiments so that the synthetase activity would


Us ---mm - , * --- U- m


A ^ A " -^ - - ^-U
Us^"* - - - u


Incubation Time (min)



Fig. 1. Fraction of original glutaminase activity remaining for hASNS after incubation time in minutes at 37�C (A), 22�C (i), and 4oC (*).
University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
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ATTEMPT TO CRYSTALLIZE HUMAN ASPARAGINE SYNTHETASE BOUND TO Two INHIBITORS


- - - - - -


10 20


30 40 50


Incubation Time (min)

Fig. 2. Fraction of original ammonia-dependent synthetase activity remaining for hASNS after incubation time in minutes at 37�C ( m), and 22�C (*).


be minimally affected. This allowed for the efficient
synthetase inhibition by sulfoximine (Fig. 4).

Inhibition ofDON-inhibited hASNS by sulfoximine
The activity of both the free and DON-inhibited
enzyme in the absence of sulfoximine remained fairly
constant throughout the 30 minutes experiment, showing
that most of the activity loss in the other reactions was due
to inhibition (Fig. 4). It took 30 minutes and 16 gM
sulfoximine to show the same loss of activity seen when
the free enzyme was incubated for 20 minutes with 10 gM
sulfoximine. The fits of these data to Equation 3 allowed
for the calculation of K, and therefore k6 values for each
reaction.
The K values from Equation 3 were plotted against
the corresponding sulfoximine concentration (Fig. 5) and
fit to Equation 5 using the average k6 value to obtain k5 and
K,. K, was found to be 285 nM (Table 1). K,* was
determined to be 2.46 nM using Equation 2.
Table 1 compares these values to those found
previously [13] for the free enzyme. Although the values
for K,* remain close, K, for free hASNS is significantly
lower than that of the DON inhibited enzyme. This reflects
the greater incubation time and concentration required to
inhibit the DON-inhibited enzyme. However, since the
values for k5 and k6 are relatively higher and lower in the
DON-inhibited enzyme, K,* is primarily unaffected.
Also, sulfoximine did not interfere with
DON inhibition in any way, both when present with
DON in the initial incubation reaction and when the Enzyr
DON-inhibited enzyme was then inhibited by the
sulfoximine. Free h


hASN:


5 10 15 20 25 30 35
Incubation Time (min)


Fig. 3. Fraction of original glutaminase activity remaining for hASNS after
incubation time in minutes in 5.88 mM DON at 22�C.




Table 1. Comparison of the values for k5, k6, K,, and K,* for sulfoximine
inhibition for the free form of hASNS [13] and the DON-inhibited form of
hASNS.



e k5 (s ') k6 (S-1) K, (nM) K,* (nM)

ASNS 2.98 x 103 2.60 x 105 285 2.46


S + DON 3.41 x 103 1.46 x 105 679 2.9


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
5


1

0.8

e 0.6

0.4
()
0.2

0





ALEXANDRIA H. BERRY ET AL.


25


20
0
U-
15

Ce
V) 10


5


120




100



0

,-
S80


o
0


c
Q.
0
40
0P


0 200 400 600 800 1000
Time (s)


Fig. 4. Concentration of pyrophosphate (pM
synthetase reaction over time in seconds at 37�C
0 (A) and 10 pM sulfoximine (o), and the DON
(A), 10 (0 ), 12 (m), 14 (*), 16 (0), and 18 pM ()
were fit to Equation 3 using KaleidaGraph.


Crystallization screens
Crystallization screens for the doubly inhibited
enzyme were performed. Crystals were obtained, but did
1200 1400 1600 not allow for a structure. During the screens, the enzyme
had a tendency to precipitate. Several screens showed that
produced during the solubility increased with increasing pH. This was
1) produced during the .
for the free enzyme with particularly apparent in the pHClear Suite screen (Fig. 6).
inhibited enzyme with 0 This suite contained several sets of the same salt conditions
sulfoximine. Data points over a range pH values. The most conditions with soluble

protein were those with Bicine at pH 9.


Discussion


0002




00015




0 0001




00005




0


Fig. 5. The K (inver
the fit in Fig. 4 to Eq
that reaction (pM). D


Since the overall goal of this work was to obtain a
crystal structure of hASNS, it was important that the
enzyme was not damaged by the actual inhibition
* * conditions. For this reason, when hASNS was inhibited
with DON, the temperature was kept at 220C, rather than
Sthe normal assay temperature of 370C.
DON inhibited hASNS quickly and irreversibly,
allowing for the preparation of milligram amounts of the
DON-inhibited enzyme, which could be stored and used
for latter studies, including sulfoximine inhibition and
crystallization screens.
The most interesting result of the inhibition
experiments was the observation that in order to achieve
the same loss of activity for the DON-inhibited enzyme as
Sfor free hASNS, a higher concentration of sulfoximine and
5 10 15 20 25
[I] (pM) a longer incubation time were required. This was reflected
ted seconds) values obtained for each reaction from in the values for K,, but not in K,*.This change in K, shows
uation 3 versus the concentration of sulfoximine [I] in that it is harder for the DON-inhibited enzyme to reach the
)ata were fit to Equation 5. initial enzyme-inhibitor complex, E.I (Scheme 1).


University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
6


0 * t I 1L I I I I I I
4.0 5.0 6.0 7.0 8.0 9.0
pH

Fig. 6. The form of the enzyme observed in the pHClear Suite
crystallization screen at various pH. Horizontal stripes represent the
numbers of conditions with precipitated protein, the solid black bars
represent the number of conditions with soluble protein, and the white
bars represent the number of conditions where another form of the
enzyme was observed, such as crystals.





ATTEMPT TO CRYSTALLIZE HUMAN ASPARAGINE SYNTHETASE BOUND TO Two INHIBITORS


However, the fact that the values for k5 and k6 are
relatively higher and lower in the DON-inhibited enzyme
means that once in the E.I form, it is easier for the enzyme
to form the tightly bound enzyme-inhibitor complex, EI*.
These two phenomena minimize the change in K,*.
These changes reflect a possible change in enzyme
conformation upon the binding of glutamine. When
glutamine is bound to the enzyme, the glutaminase reaction
occurs and ammonia is released down the tunnel to the C-
terminal active site. In this case, the enzyme must be ready
to proceed with the synthetase reaction to avoid fruitless
glutamine breakdown. Therefore, it is possible that the
enzyme would partly close-up its synthetase site to keep
any substrates from escaping. In this experiment, DON
acts as glutamine does by binding to the glutaminase site.
This act of mimicking the glutaminase reaction may cause
the enzyme to partially close-up its synthetase active site to
prepare for the reaction. A constricted C-terminal active
site would make it more difficult for the sulfoximine to
bind, but once bound, it might facilitate the formation of
the tightly-bound complex.
Although no crystal structure was obtained,
valuable information regarding solubility was discovered.
hASNS was found to be more soluble at higher pH. This
information has been used to develop new storage and
purification conditions for the expression of hASNS.
Bicine at pH 8.7 and 100 mM NaCl is now used to store
the enzyme instead of EPPS at pH 8. This has allowed for
increased solubility, allowing the enzyme to be more
concentrated, an important goal of crystallography.
Because of these studies, it is possible to prepare
milligram amounts of soluble, doubly-inhibited enzyme.
The inhibition of hASNS by sulfoximine may have
revealed an aspect of its reaction mechanism, one which
involves a conformational change. This information will
hopefully help facilitate future attempts to gain a crystal
structure of hASNS, so that rational approaches can be
used to design hASNS inhibitors able to treat drug-resistant
leukemia.

Acknowledgements

I would like to thank Dr. Kai Li, for teaching me
how to conduct the enzyme assays, for his help performing
experiments, and his advice on experimental design.
Thank you also to Cory Toyota and Megan Myer for
teaching me how to use many of the instruments in the lab,
and helping with enzyme purification. I would like to
thank Dr. Inari Kursula at the Karolinska Institutet for
working with me on the screens, and her advisor, Dr. Ylva
Lindqvist, who allowed me to come and work in her lab. I
would also like to thank Dr. Richards, who has been an
excellent advisor, for allowing me to work on the project.


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University of Florida I Journal of Undergraduate Research I Volume 10, Issue 3 I Spring 2009
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