PMID:9890911
| Citation |
Deras, ML, Chittur, SV and Davisson, VJ (1999) N2-hydroxyguanosine 5'-monophosphate is a time-dependent inhibitor of Escherichia coli guanosine monophosphate synthetase. Biochemistry 38:303-10 |
|---|---|
| Abstract |
In contrast to several other glutamine amidotransferases including asparagine synthetase, cytidine 5'-triphosphate (CTP) synthetase, carbamoyl phosphate synthetase, and phosphoribosyl pyrophosphate (PRPP) amidotransferase, guanosine monophosphate synthetase (GMPS) will not utilize hydroxylamine as an alternative nitrogen source. Instead, the enzyme is inhibited by an unknown mechanism. One untested hypothesis was that hydroxylamine serves as a substrate and intercepts a xanthosine 5'-monophosphate- (XMP-) adenylate intermediate in the enzyme active site. The nucleotide product of this substitution reaction would be N2-hydroxyguanosine 5'-monophosphate (N2-OH-GMP, 2). Here we describe the chemoenzymatic preparation of 2, via the nucleotide 2-fluoroinosine 5'-monophosphate (F-IMP, 5), and characterization of both these compounds as inhibitors of Escherichia coli GMPS. F-IMP was conceived as an electronic mimic of a reactive intermediate in the GMPS reaction but was found to bind weakly to the enzyme (IC50 > 2 mM). In contrast, N2-OH-GMP shows time-dependent inhibition and is competitive with respect to XMP (Ki = 92 nM), representing the first example of a compound that displays these kinetic properties with GMPS. The mechanism of inhibition is proposed to occur via formation of a ternary E.ATP.2 complex, followed by a rate-determining isomerization to a higher affinity complex that has a t1/2 =7.5 min. The contrast in inhibitory activity for 2-substituted purines with GMPS formulates a basis for future inhibitor design. In addition, these results complement recent structural studies of GMPS and implicate the formation of the XMP-adenylate intermediate inducing a probable conformational change that stimulates the hydrolysis of glutamine. |
| Links |
PubMed Online version:10.1021/bi981980r |
| Keywords |
Adenosine/analogs & derivatives; Adenosine/pharmacology; Adenosine Triphosphate/chemistry; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/antagonists & inhibitors; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/chemistry; Enzyme Activation/drug effects; Enzyme Inhibitors/pharmacology; Glutaminase/chemistry; Guanosine Monophosphate/analogs & derivatives; Guanosine Monophosphate/pharmacology; Hydrolysis; Kinetics; Models, Chemical |
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