Enzymatic Mechanisms of Sulfur Nucleoside Metabolism

说明（由申请人提供）S-腺苷甲硫氨酸（S-腺苷甲硫氨酸）在所有细胞生物体的代谢中起重要作用，其功能通常在疾病中改变。代谢的酶是开发化疗剂的靶标。本研究的目的是阐明S-腺苷甲硫氨酸合成酶（ATP：L-甲硫氨酸S-腺苷转移酶，MAT）和蛋氨酸脱羧酶（蛋氨酸脱羧酶）的功能，其反应使蛋氨酸多胺合成。大量的实验和计算技术将提供有关酶功能的新的结构和机制信息，并导致发现新的抑制剂。 MAT的机制将通过计算方法来表征，以阐明 催化这两步反应。实验数据指导量子力学在 研究活性位点残基的作用。机械论的推论将在实验中得到检验。 新型MAT抑制剂将通过小分子库与MAT晶体结构的虚拟对接来发现。候选抑制剂将通过实验进行评价，以鉴定能够调节细胞CD 3 Met水平的化合物。人类病原体化脓性链球菌具有通常仅在古细菌中发现的MAT类型和细菌形式。假设古细菌类型MAT使用除了ATP之外的底物合成新的代谢物;这可以提供新的抗生素靶标。克隆了S.化脓菌MAT将在E.大肠杆菌，纯化和表征。如果特异性假说在体外得到证实，S.化脓性链球菌将揭示这些新的代谢物是否在体内形成。将对比两种含有异戊酰辅因子的异戊酰MetDC的明显不同的催化机制。动力学和结构研究将阐明活化剂独立类的代表和金属离子依赖组的代表的机制。蛋白质（和金属离子）对辅因子和酶底物复合物的电子环境的影响将从选择性富集的蛋白质和底物的复合物的13 C和15 N NMR中揭示。这些研究将揭示为什么自然界保留使用一种异戊酰辅因子来实现这种代谢功能，而不是采用常见的吡哆醛辅因子。

DESCRIPTION (provided by applicant) S-adenosylmethionine (AdoMet) plays essential roles in the metabolism of all cellular organisms, with functions that are commonly altered in diseases. The enzymes of AdoMet metabolism are targets for development of chemotherapeutic agents. The goals of this research are to elucidate the functioning of S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, MAT), and of AdoMet decarboxylase (AdoMetDC) whose reaction commits AdoMet to polyamine synthesis. A plethora of experimental and computational techniques will provide new structural and mechanistic information regarding enzyme function, and lead towards discovery of novel inhibitors. The mechanism of MAT will be characterized by computational methods to elucidate the basis of catalysis of this two-step reaction. Experimental data guide the use of quantum mechanics in investigation of the roles of active site residues. Mechanistic deductions will be tested experimentally. Novel MAT inhibitors will be discovered by virtual docking of libraries of small molecules to the MAT crystal structure. Candidate inhibitors will be experimentally evaluated to identify compounds able to modulate cellular AdoMet levels. The human pathogen Streptococcus pyogenes has both a type of MAT typically found only in archaea and the bacterial form. The archaeal type MAT is hypothesized to synthesize novel metabolites using substrates in addition to ATP; this could provide a new antibiotic target. Both cloned S. pyogenes MATs will be expressed in E. coli, purified and characterized. If the specificity hypothesis is confirmed in vitro, extracts of S. pyogenes will reveal if these novel metabolites form in vivo. The apparently diverse catalytic mechanisms of two non-homologous pyruvoyl cofactor containing AdoMetDC will be contrasted. Kinetic and structural studies will elucidate the mechanisms of a representative of the activator independent class and a representative of the metal ion dependent group. The influence of the protein (and metal ion) on the electronic environment of the cofactor and the enzyme substrate complexes will be revealed from 13C and 15N NMR of complexes of selectively enriched protein and substrates. These studies will unmask why nature conserves use of a pyruvoyl cofactor for this metabolic function instead of adopting the common pyridoxal cofactor.