A potent transition-state analogue inhibitor of Escherichia coli asparagine synthetase A

Full text of DOI:10.1021/ja990851a

J. Am. Chem. Soc. 1999, 121, 5799-5800
5799
Scheme 1
A Potent Transition-State Analogue Inhibitor of
Escherichia coli Asparagine Synthetase A
Mitsuteru Koizumi, Jun Hiratake,* Toru Nakatsu,
Hiroaki Kato, and Jun’ichi Oda†
Institute for Chemical Research, Kyoto UniVersity
Uji, Kyoto 611-0011, Japan
Department of Bioscience
Fukui Prefectual UniVersity, Matsuoka-cho
Yoshida-gun, Fukui 910-1195, Japan
ReceiVed March 16, 1999
L-Asparagine synthetase [L-aspartate: ammonia ligase (AMP-
forming) EC 6.3.1.1] (AS-A) from Escherichia coli1 is a typical
member of ammonia-dependent asparagine synthetases2 and
catalyzes the formation of L-Asn from L-Asp and ammonia with
concomitant hydrolysis of ATP to AMP and pyrophosphate. In
asparagine biosynthesis, another family of asparagine synthetases
(AS-B) which utilize glutamine as a nitrogen source is more
ubiquitous for eukaryotes.3 Asparagine synthetase is a potential
target for cancer chemotherapy3 because asparagine depletion
caused by the administration of L-asparaginase is a currently
implemented protocol for the treatment of acute lymphoblastic
leukemia.4 Although AS-A is not related structurally and evolu-
tionarily to AS-B3 and is more related to amino-acyl tRNA
synthetases,5 the reaction catalyzed by AS-A is prototypic of this
class of enzyme from a mechanistic point of view: the substrate
carboxyl group is activated by adenylation followed by substitu-
tion by amine nucleophile.2a,b,6 It is therefore highly desirable to
obtain good inhibitors of AS-A not only for use as a probe to
define the detailed reaction mechanisms of asparagine synthetases,
but also for generating a lead for chemotherapeutic agents targeted
toward the inhibition of asparagine biosynthesis. For these
purposes, transition-state analogues7 are far more suitable than
substrate analogues8 or intermediate mimics.9 We now describe
the synthesis and characterization of a transition-state analogue,
N-adenylated S-methyl-L-cysteine sulfoximine 1. Compound 1
served as an extremely potent slow-binding inhibitor of E. coli
AS-A with an overall inhibition constant of 67 nM. An X-ray
crystal structure of the enzyme complexed with 1 revealed several
key amino acid residues responsible for catalysis as well as those
for substrate recognition.
Scheme 2a
a Reagents and conditions: (a) NaIO4, MeOH-H2O, 25 °C, 7 h; (b)
O-mesitylsulfonylhydroxylamine (MSH), CH3CN, 25 °C, 3 days; (c) 2′,3′-
O-isopropylideneadenosine, diisopropylammonium 1H-tetrazolide, dry
pyridine, 25 °C, 15 h; (d) 1H-tetrazole, dry CH3CN, 25 °C, 4 h, then
t-BuOOH; (e) TFA-H2O (8:3), 25 °C, 11 h; (f) Pd(PPh3)4, PPh3, HCOOH,
NEt3, dry THF, 25 °C, 9 h; (g) H2, 10% Pd-C, CH3CN-H2O, 1 h.
The catalytic reaction of AS-A is thought to proceed by a two-
step mechanism involving an intermediate â-aspartyl adenylate;2a,b
the first step is the formation of the intermediate, and the
adenylated â-carboxyl is attacked by ammonia in the second step
to form L-Asn and AMP (Scheme 1). As a stable analogue of the
transition state in the latter step, we designed N-adenylated
S-methyl-L-cysteine sulfoximine 1 where the carbonyl to be
attacked by ammonia is replaced by a tetrahedral sulfoximine
sulfur atom with a methyl group mimicking ammonia. The
synthesis of 1 is shown in Scheme 2. The sulfoximine nitrogen
was adenylated by a conventional phosphoramidite method,10 and
the resulting P-N bond was hydrolytically stable even under
acidic conditions. Compound 1 was obtained as a 1:1 mixture of
diastereomers with respect to the chiral sulfur atom.
The N-adenylated sulfoximine 1 was found to be a potent slow-
binding inhibitor that caused time-dependent inactivation of AS-A
(Figure 1). The enzyme, for example, was totally inactivated in
15 min when 2.5 µM of 1 was present. The inhibition was virtually
irreversible, and no regain of enzyme activity was observed after
gel filtration.11 Since the inhibition was time-dependent, the onset
† Fukui Prefectual University.
(1) (a) Sugiyama, A.; Kato, H.; Nishioka, T.; Oda, J. Biosci. Biotechnol.
Biochem. 1992, 56, 376-379. (b) Hinchman, S. K.; Schuster, S. M. Protein
Eng. 1992, 5, 279-283.
(2) (a) Cedar, H.; Schwartz, J. H. J. Biol. Chem. 1969, 244, 4112-4121.
(b) Cedar, H.; Schwartz, J. H. J. Biol. Chem. 1969, 244, 4122-4127. (c) Kim,
S. Il; Germond, J.-E.; Pridmore, D.; So¨ll, D. J. Bacteriol. 1996, 178, 2459-
2461.
(3) Richards, N. G. J.; Schuster, S. M. In AdVances in Enzymology and
Related Areas of Molecular Biology; Purich, D. L., Ed.; John Wiley: New
York, 1998; Vol. 72: Amino Acid Metabolism, Part A, pp 145-198 and
references therein.
(4) Mu¨ller, H. J.; Boos, J. Crit. ReV. Oncol. Hematol. 1998, 28, 97-113.
(5) (a) Nakatsu, T.; Kato H.; Oda, J. Nat. Struct. Biol. 1998, 5, 15-19. (b)
Hinchman, S. K.; Henikoff, S.; Schuster, S. M. J. Biol. Chem. 1992, 267,
144-149. (c) Gatti, D. L.; Tzagoloff, A. J. Mol. Biol. 1991, 218, 557-568.
(6) Luehr, C. A.; Schuster, S. M. Arch. Biochem. Biophys. 1985, 237, 335-
346.
(7) (a) Wolfenden, R. Annu. ReV. Biophys. Bioeng. 1976, 5, 271-306. (b)
Radzicka, A.; Wolfenden, R. Methods Enzymol. 1995, 249, 284-312. (c)
Schramm, V. L. Annu. ReV. Biochem. 1998, 67, 693-720.
(8) Several aspartic acid analogues were synthesized as potential inhibitors
of asparagine synthetase B (Parr, I. B.; Boehlein, S. K.; Dribben, A. B.;
Schuster, S. M.; Richards, N. G. J. J. Med. Chem. 1996, 39, 2367-2378 and
references therein).
(9) (a) Pike, D. C.; Beevers, L. Biochim. Biophys. Acta 1982, 708, 203-
209. (b) Zhukov, Yu. N.; Biryukov, A. I.; Khomutov, R. M. Bioorg. Khim.
1988, 14, 969-972 (Chem. Abstr. 1988, 109, 207322w).
(10) The amino group of adenine ring was unreactive under tetrazole- or
tetrazolium salt-catalyzed phosphinylation conditions, and no protection was
necessary.
(11) The enzyme did not regain activity for 10 days after gel filtration, but
a very slow regain of enzyme activity was observed in the absence of Mg2+
.
10.1021/ja990851a CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/05/1999

5800 J. Am. Chem. Soc., Vol. 121, No. 24, 1999
Communications to the Editor
Figure 1. Progress curves for the inactivation of AS-A by transition-
state analogue 1. The reaction was initiated by adding the enzyme to an
assay mixture containing L-Asp (1.5 mM), NH4Cl (20 mM), ATP (3 mM)
and the inhibitor 1 (0-7.5 µM) in 100 mM Tris-HCl (pH 7.8) at 37 °C.
Figure 2. X-ray crystal structure of E. coli AS-A active site complexed
with the inhibitor 1, showing the SIGMAA weighted Fo - Fc omit
electron density map (blue), superimposed with the refined structure of
the inhibitor 1 and the active site amino acid residues. The contour level
is 3.0 σ.
rate of inactivation (kon)12 was calculated from the progress curves
and was found to be 3.27 s-1 mM-1 13
The overall inhibition
.
constant (Ki*)12 was calculated as 67 nM by measuring the
inhibited enzyme activity after slow-binding inhibition equilibrium
was reached. Considering that the kinetic constants for AS-A were
Km (Asp) ) 1.69 mM, Km (ATP) ) 1.24 mM, and k0/Km (Asp)
) 28.5 s-1 mM-1, the inhibitor 1 binds to the enzyme at a rate 9
times smaller than that of the enzymatic reaction, but with 25000-
fold higher affinity than its substrates.
We first attempted to inhibit AS-A with diastereomeric
S-methyl-L-cysteine sulfoximine 6a,b14 and ATP in the hope that
a mechanism-based inactivation of AS-A might result by in situ
adenylation of the sulfoximine nitrogen by ATP within the enzyme
active site. However, each diastereomer 6a and 6b was a poor
and reversible inhibitor of AS-A15 with Ki ) 3.27 and 0.76 mM,
respectively, and no enzyme inactivation was observed after a
prolonged incubation of the enzyme with 6a or 6b in the presence
of ATP. Since chemically synthesized 1 served as an extremely
potent inhibitor of AS-A, the enzyme was not capable of
adenylation of the sulfoximine 6. This result is in sharp contrast
to the inhibition of another class of ATP-dependent ligases such
as glutamine synthetase16 and γ-glutamylcysteine synthetase17 by
sulfoximine-based transition-state analogues, where the sulfox-
imine nitrogen was phosphorylated in a mechanism-based manner
to cause time-dependent inactivation of the enzyme.
and subjected to X-ray diffraction analysis. The inhibitor 1 gave
a clear electron density in the enzyme active site, and several
specific interactions were identified between the active-site amino
acid residues and the inhibitor 1 (Figure 2). Of particular interest
is the interaction regarding the sulfoximine moiety. First, the
sulfoximine oxygen (SdO) is hydrogen bonded to Arg 100 and
Gln 116. Since SdO represents the oxyanion generated by
nucleophilic attack of ammonia, these residues are most likely to
be key catalytic residues responsible for stabilizing the tetrahedral
oxyanion transition state or intermediate. Another point is the
methyl attached to the sulfur atom. This methyl, which is supposed
to mimic the -NH3+ moiety of the zwitterionic tetrahedral
transition state or intermediate, was found to be 3.1 Å distant
from Asp 46. This negatively charged residue probably plays a
key role in electrostatic stabilization of the -NH3+ part of the
transition state and also acts as a base to abstract a proton from
the tetrahedral intermediate completing the formation of the
product asparagine.18
In conclusion, a potent slow-binding inhibitor 1 of E. coli
asparagine synthetase A was prepared. The crystal structure of
the enzyme complexed with 1 gave a structural basis for the
transition-state stabilization as well as for substrate recognition
by the enzyme. Since this enzyme shares the same chemistry in
carboxylate activation with glutamine-dependent asparagine syn-
thetases (AS-B), and all AS-B from eukaryote can employ
ammonia as an alternative nitrogen source in asparagine synthe-
sis,3 compound 1 should also formulate a basis for future inhibitor
design of asparagine synthetase B.
Since compound 1 was most likely to mimic the putative
transition state, the enzyme complexed with 1 was crystallized
(12) Morrison, J. F.; Walsh, C. T. In AdVances in Enzymology; Meister,
A., Ed.; John Wiley: New York, 1988; Vol. 61, pp 201-301. The kon value
was calculated from a plot of pseudo-first-order inhibition rates (kobs) vs [1]
by regression to kobs ) kon [1] since no saturation was observed. Ki* was
obtained by steady-state inhibition kinetics after the enzyme was preincubated
with various concentrations of 1 (0-7.5 µM) at 37 °C for 30 min to establish
the binding equilibrium. The substrate concentrations are [Asp] ) 1.5 mM,
[ATP] ) 3 mM and [NH4+Cl-] ) 20 mM. The initial inhibition constant (Ki)
was not obtained because the plot of kobs vs [1] showed no saturation.
(13) We examined the effect of pyrophosphate (PPi) to see if the generation
and binding of PPi would be a possible cause for time-dependent inactivation
of the enzyme, but no effect was observed on the rate or the extent of enzyme
inactivation up to 10 mM of PPi.
Acknowledgment. This study was supported in part by a Grant-in-
Aid for Scientific Research from the Ministry of Education, Science,
Sports and Culture of Japan to which we are grateful. We thank Ms.
Yuka Horiuchi and Ms. Yukiji Kitamura for their assistance in the
synthesis of sulfoximine 6 and the inhibitor 1, respectively.
(14) The sulfoximine 6 was synthesized from S-methyl-L-cysteine by
stepwise oxidation with NaIO4 and HN3/H2SO4 by modifying the reported
procedure (Griffith, O. W. Methods Enzymol. 1981, 77, 59-64). The
diastereomers 6a and 6b with respect to the chiral sulfur were separated by
fractional crystallization from EtOH-H2O.
Supporting Information Available: Experimental procedures (PDF).
This material is available free of charge via the Internet at http://pubs.acs.org.
JA990851A
(15) L-Cysteine sulfoximine was reported to be a poor inhibitor of
glutamine-dependent AS-B from mammalian pancreas (Jayaram, H. N.;
Cooney, D. A. Cancer Treat. Rep. 1979, 63, 1095-1108).
(18) A similar role was reported for Glu 327 of E. coli glutamine synthetase;
+
(16) Rowe, W. B.; Ronzio, R. A.; Meister, A. Biochemistry 1969, 8, 2674-
2680.
(17) (a) Tokutake, N.; Hiratake, J.; Katoh, M.; Irie, T.; Kato, H.; Oda, J.
Bioorg. Med. Chem. 1998, 6, 1935-1953. (b) Griffith, O. W. J. Biol. Chem.
1982, 257, 13704-13712.
the carboxyl group of Glu 327 was within 2.6 Å of the location of the -NH3
moiety of the tetrahedral intermediate and was shown to be responsible for
stabilizing the transition state (Liaw, S.-H.; Eisenberg, D. Biochemistry 1994,
33, 675-681. Alibhai, M.; Villafranca, J. J. Biochemistry 1994, 33, 682-
686).