Inhibition of Guanosine Monophosphate Synthetase by the Substrate Enantiomer L-XMP

Full text of DOI:10.1002/cbic.201200503

DOI: 10.1002/cbic.201200503
Inhibition of Guanosine Monophosphate Synthetase by the Substrate
Enantiomer l-XMP
Nicholas B. Struntz, Tianshun Hu, Brian R. White, Margaret E. Olson, and Daniel A. Harki*[a]
Studies with mirror-image l-enantiomer nucleosides and nucle-
otides have revealed relaxed enantioselectivities of several cel-
lular kinases and viral polymerases.[1,2] This feature of enzyme–
ligand molecular recognition has been exploited in the design
of efficacious antiviral l-nucleoside drugs, which have lowered
host-cell toxicity.[3–6] For example, lamivudine (2’,3’-dideoxy-3’-
thiacytidine, 3TC), an l-nucleoside drug, exploits the relaxed
enantioselectivity of human immunodeficiency virus (HIV) re-
verse transcriptase to inhibit viral replication.[7] Conversely, the
enantioselectivities of the majority of nucleotide biosynthesis
enzymes have not been characterized. The depletion of cellular
nucleotide pools has been shown to result in antiproliferative,
antibacterial, and immunosuppressive effects.[8–11]
target GMPS and modulate enzymatic activity. We hypothe-
sized that l-XMP could incorporate into the synthetase active
site and inhibit enzyme function, or less likely, l-XMP could
function as a substrate for GMPS and undergo aminolysis to
yield l-GMP. In either case, enzymatic synthesis of d-GMP
would be affected, either by direct enzyme inhibition or by the
activity of a suicide substrate. Given the central importance of
GMPS in eukaryote and prokaryote biochemistry, we examined
the enantioselectivity of the enzyme.
Preceding this work, a synthesis of l-XMP (6), the enantio-
mer of natural ligand d-XMP, had not been reported. Our syn-
thesis of l-XMP (6) started from l-arabinose, which was elabo-
rated to 1-O-acetyl-2,3,5-tri-O-benzoyl-b-l-ribofuranoside (1) by
reported methods (Scheme 1).[19] A Vorbrꢁggen coupling with
Guanosine monophosphate synthetase (GMPS), an enzyme
involved in de novo nucleotide biosynthesis, catalyzes the ami-
nation of xanthosine 5’-monophosphate (XMP) to guanosine
5’-monophosphate (GMP) in the presence of glutamine (the
amine source) and ATP.[10,12] GMPS possesses two active sites
that are separated by approximately 30 ꢀ, thus suggesting
that GMPS undergoes a significant conformational change
during catalysis.[13,14] In the amidotransferase active site, a gluta-
mine residue is hydrolyzed to liberate ammonia, which subse-
quently functions as the nucleophile in the amination of XMP
[Eq. (1)]. In the synthetase active site, the 2-carbonyl of XMP is
adenylated with ATP [Eq. (2)] to activate the aromatic ring for
subsequent aminolysis [Eq. (3)]. Formation of adenyl-XMP is
believed to trigger glutamine hydrolysis in the amidotransfer-
ase active site.[10,12–16]
glutamine þ H O GMPS glutamate þ NH
ð1Þ
ð2Þ
ð3Þ
ƒƒ!
2
3
GMPS
XMP þ ATP
adenyl ꢀ XMP þ PPi
G
H
Scheme 1. Synthesis of l-XMP. Reagents and conditions: a) TMSOTf, CH2Cl2,
reflux, 70% (for 3), 21% (for N7-isomer 4, not shown); b) NH3, MeOH, 558C
(sealed tube), 93%; c) POCl3, PO(OMe)3, proton sponge; aq TEAB, 19%.
GMPS
adenyl ꢀ XMP þ NH3
GMP þ AMP
ƒƒ!
A crystal structure of Escherichia coli GMPS has been solved
that reveals a large solvent-accessible synthetase pocket with
considerable surface area.[13] Several base-modified d-XMP ana-
logues have been shown to function as substrates for GMPS
and be converted to their amine derivatives,[17] and non-hydro-
lyzable adenyl-XMP analogues have been synthesized.[18] Based
on this structural information and our interest in characterizing
for the first time the enantioselectivity of GMPS, we hypothe-
sized that l-XMP, the enantiomer of native ligand d-XMP, could
trimethylsilyl-protected xanthine 2 gave a separable mixture of
protected l-xanthosine isomers 3 (N9 isomer) and 4 (N7 isomer,
not shown).[20,21] Deprotection of the benzoyl protecting
groups of 3 by using ammonia afforded l-xanthosine (5). Se-
lective phosphorylation of the 5’-OH of 5 utilizing phosphorous
oxychloride gave l-XMP (6).[22]
E. coli GMPS was overexpressed and purified (Figure S1) and
an HPLC-based assay was developed to quantitate enzymatic
reaction products. GMPS was incubated with test substrates
and ammonium acetate (ammonia source), and the reaction
was terminated at various time points by addition of ethylene-
diaminetetraacetic acid (EDTA). GMPS protein was then re-
moved by a molecular weight spin column (30 kDa), and enzy-
matic reaction products were analyzed by reversed-phase
[a] N. B. Struntz,+ Dr. T. Hu,+ Dr. B. R. White, M. E. Olson, Prof. Dr. D. A. Harki
Department of Medicinal Chemistry, University of Minnesota
717 Delaware Street S.E., Minneapolis, MN 55414 (USA)
E-mail: daharki@umn.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200503.
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Analysis of d-XMP revealed
an apparent Km value of
35.3 mm, which was similar to
previously reported Km values
for E. coli GMPS (29 mm and
166 mm).[23,24]
The
turnover
number (kcat) was found to be
4.8ꢃ10ꢀ2 sꢀ1, which was compa-
Figure 1. HPLC analysis of l-XMP conversion to l-GMP by GMPS.
rable to a previous report of
HPLC. Surprisingly, we found that incubation of l-XMP and
GMPS yielded a new peak of identical retention time as d-GMP
(Figure 1). Characterization of this new peak (MS analysis, Fig-
ure S3) confirmed that l-XMP was converted to l-GMP by
GMPS, demonstrating that turnover of the opposite enantio-
mer substrate was possible.
9.4ꢃ10ꢀ2 sꢀ1 [24]
.
Analysis of l-XMP revealed an apparent Km
value of 316.7 mm, which is approximately ten times higher
than that of the natural enantiomer. Surprisingly, the kcat value
was measured at 3.2ꢃ10ꢀ6 sꢀ1, which is a 15000-fold difference
in turnover number compared with d-XMP. The specific activity
(kcat/Km) of l-XMP decreased 140000-fold from the natural
enantiomer (1.4ꢃ10ꢀ3 mmꢀ1 sꢀ1 for d-XMP versus 1.0ꢃ
10ꢀ8 mmꢀ1 sꢀ1 for l-XMP). These results suggest that l-XMP
might also inhibit GMPS.
Biochemical characterization of the kinetics of l-XMP conver-
sion to l-GMP by GMPS, as well as d-XMP conversion to d-
GMP, was measured by fitting the individual GMP/cytosine 5’-
monophosphate (CMP, an external standard) ratios from each
sample into the slope-intercept equation from the calibration
plot. Initial velocity measurements of GMP production as
a function of time were measured at a variety of substrate
(XMP) concentrations, and at fixed saturating concentrations of
the non-varied substrates ATP and ammonium acetate (Fig-
ure 2A). Fitting these data to the Michaelis–Menten equation
and analysis by nonlinear regression allowed measurement of
kinetic parameters (Table 1).
To confirm the values derived from the HPLC assay, a known
continuous UV spectrophotometric assay was also em-
ployed.[23,25] This assay monitors a reduction in 290 nm absorb-
ance resulting from conversion of XMP (e290 =4800mꢀ1 cmꢀ1) to
GMP (e290 =3300mꢀ1 cmꢀ1). UV-based kinetic data was calculat-
ed analogously to the HPLC-derived data (Figure 2B). Analysis
of both d-XMP and l-XMP revealed nearly identical results to
the HPLC assay (for l-XMP: kcat/Km =9.4ꢃ10ꢀ9 mmꢀ1 sꢀ1 (UV)
versus 1.0ꢃ10ꢀ8 mmꢀ1 ꢀ1 (HPLC); Table 1).
s
Although l-XMP conversion to l-GMP by GMPS was demon-
strated, the weak affinity of l-XMP for GMPS, coupled with its
slow turnover number, suggested possible enzyme inhibition
by this ligand. To probe for GMPS inhibition, we performed en-
zymatic activity experiments with our xanthosine-based mole-
cules to demonstrate reduction of GMPS-mediated amination
of d-XMP. Addition of a fixed concentration of inhibitor to vary-
ing d-XMP concentrations, followed by UV–visible analysis and
fitting to the competitive inhibition equation (or uncompeti-
tive for decoyinine towards XMP),[10] yielded the Ki data shown
in Table 2. Evaluation of the known GMPS uncompetitive inhib-
Table 2. GMPS inhibition by known inhibitors and xanthosine ana-
logues.[a]
Inhibitor
Ki [mm]
Inhibitor
Ki [mm]
1.8ꢁ0.7
decoyinine
d-xanthosine
l-XMP
54.1ꢁ14.5
mizoribine
l-xanthosine
>1500
7.5ꢁ1.8
>500
Figure 2. Initial velocities versus substrate concentration plots for d-XMP
and l-XMP as ligands for GMPS as measured by A) HPLC analysis and B) UV/
Vis analysis.
[a] Performed in triplicate. Values shown are the meanꢁSD.
Table 1. Kinetic parameters of GMPS enzymatic activity in the presence
of enantiomeric substrates d-XMP and l-XMP.[a]
itor decoyinine revealed a Ki value of 54.1 mm, which was simi-
lar to a previous report (26 mm).[26] Mizoribine (bredinin trade
name), a known immunosuppresive drug and competitive
GMPS inhibitor, was found to be more potent in our hands
(Ki =1.8 mm); this activity is similar to reports of the same com-
pound against GMPS isolated from rat Walker sarcoma cells
(Ki =10 mm).[11] l-XMP (6) inhibition results were quite interest-
ing, revealing that l-XMP is almost seven times more potent
Substrate
kcat [sꢀ1
]
Km [mm]
kcat/Km [mmꢀ1 sꢀ1
]
d-XMP[b]
d-XMP[c]
l-XMP[b]
l-XMP[c]
(4.8ꢁ0.3)ꢃ10ꢀ2
(4.3ꢁ0.7)ꢃ10ꢀ2
(3.2ꢁ0.3)ꢃ10ꢀ6
(3.1ꢁ0.7)ꢃ10ꢀ6
35.3ꢁ8.5
24.9ꢁ6.6
1.4ꢃ10ꢀ3
1.7ꢃ10ꢀ3
1.0ꢃ10ꢀ8
9.4ꢃ10ꢀ9
316.7ꢁ55.6
329.9ꢁ104.9
[a] Performed in triplicate. Values shown are the meanꢁSD. [b] HPLC
analysis. [c] UV/Vis analysis.
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than decoyinine inhibition against E. coli GMPS (Ki =7.5 mm).
Both d-xanthosine and l-xanthosine nucleosides were also
tested and neither molecule inhibited GMPS; this suggests
that 5’-monophosphorylation is required for inhibition. Mizori-
bine does not require phosphorylation for GMPS inhibition.
Our results suggest that l-XMP can inhibit GMPS enzymatic ac-
tivity with potency similar to or slightly better than other
known inhibitors.[11,26–28]
tially different position within the synthetase domain (Fig-
ure 3B). No longer present were many of the key molecular
interactions between the nucleobase and ribose alcohols as
evident by a decreased consensus score, which is an estimate
of the overall ligand binding affinity (CScore=7.68 for d-XMP
versus 6.16 for l-XMP). One compensating molecular inter-
action was observed for adenyl-l-XMP, which was a hydrogen
bond between Asn761 to a ribose alcohol. The conformation
of the l-ribose sugar in adenyl-l-XMP also forces C2 of the nu-
cleobase to be positioned approximately 2.0 ꢀ away from the
region in space occupied by the natural adenyl-d-XMP ligand.
This perturbation to nucleobase conformation may deter ami-
nolysis of the adenylated unnatural monophosphate, thereby
slowing enzyme turnover. Additionally, the loss of key hydro-
gen-bonding interactions may also contribute to the loss in
enzyme efficiency. Nonetheless, our observation of the synthe-
sis of l-GMP from l-XMP implies that the large size of the syn-
thetase pocket must allow some movement of adenyl-l-XMP
to obtain the correct conformation for amination.
To understand the molecular interactions of d-XMP and l-
XMP within the GMPS active site, energy minimized three-di-
mensional conformations of the biochemical reaction inter-
mediates (adenyl-d-XMP and adenyl-l-XMP; Figure S4B) were
docked into the crystal structure of E. coli GMPS (Surflex-dock
in the SYBYL software suite; PDB ID: 1GPM[13]). The molecule of
AMP observed in the X-ray crystal structure was extracted and
re-docked into GMPS with a calculated similarity of 0.908 (1.0
is the theoretical maximum), showing reliability in docking
accuracy (Figure S4A). Several stabilizing molecular interactions
were observed between adenyl-d-XMP and GMPS, such as hy-
drogen bonds between Lys856 and the xanthine nucleobase;
Arg875, Arg765, and Glu768 to ribose alcohols; and Asn761 to
the phosphate of d-XMP (Figure 3A). The considerable size of
the GMPS pocket readily accommodated the docking of
adenyl-l-XMP; however, the l-ribose sugar occupied a substan-
In conclusion, the synthesis of l-GMP from l-XMP provides
new insight into the substrate promiscuity of GMPS. GMPS was
also inhibited by l-XMP at low micromolar levels; this is com-
parable to the behavior of other known inhibitors. These re-
sults provide new insight into GMPS–ligand interactions that
will be useful for future inhibitor designs.
Acknowledgements
This work was financially supported by an Engebretson Drug
Design & Development Grant from the Elmer and Ethel Engebret-
son Family Charitable Trust and by the University of Minnesota
(start-up funds to D.A.H.). N.B.S. and M.E.O. thank the NIH for
predoctoral traineeships (T32-GM08700). M.E.O. acknowledges
a Bighley Graduate Fellowship. We gratefully acknowledge Prof.
Janet Smith (University of Michigan) for the E. coli GMPS expres-
sion vector and Prof. Courtney Aldrich (University of Minnesota,
Center for Drug Design) for helpful discussions. We also thank
the Minnesota Supercomputing Institute (MSI) for computing re-
sources.
Keywords: enzyme catalysis · enzyme enantioselectivity ·
enzyme inhibitors · guanosine monophosphate synthetase ·
kinetics
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