Deras M L, Chittur S V, Davisson V J
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333, USA.
Biochemistry. 1999 Jan 5;38(1):303-10. doi: 10.1021/bi981980r.
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.
与包括天冬酰胺合成酶、胞苷5'-三磷酸（CTP）合成酶、氨甲酰磷酸合成酶和磷酸核糖焦磷酸（PRPP）酰胺转移酶在内的其他几种谷氨酰胺酰胺转移酶不同，鸟苷单磷酸合成酶（GMPS）不会利用羟胺作为替代氮源。相反，该酶会通过未知机制受到抑制。一个未经检验的假说是，羟胺作为底物，在酶活性位点拦截黄苷5'-单磷酸-（XMP-）腺苷酸中间体。这种取代反应的核苷酸产物将是N2-羟基鸟苷5'-单磷酸（N2-OH-GMP，2）。在这里，我们描述了通过核苷酸2-氟肌苷5'-单磷酸（F-IMP，5）化学酶法制备2，并将这两种化合物表征为大肠杆菌GMPS的抑制剂。F-IMP被设想为GMPS反应中反应中间体的电子模拟物，但发现它与该酶的结合较弱（IC50>2 mM）。相比之下，N2-OH-GMP表现出时间依赖性抑制，并且相对于XMP具有竞争性（Ki = 92 nM），这代表了一种与GMPS表现出这些动力学特性的化合物的首个实例。抑制机制被认为是通过形成三元E.ATP.2复合物，随后进行限速异构化，形成半衰期为7.5分钟的更高亲和力复合物。GMPS对2-取代嘌呤的抑制活性差异为未来抑制剂设计奠定了基础。此外，这些结果补充了最近关于GMPS的结构研究，并暗示XMP-腺苷酸中间体的形成诱导了可能的构象变化，从而刺激谷氨酰胺的水解。
