Enzymatic Mechanisms of Sulfur Nucleoside Metabolism

S-腺苷甲硫氨酸(ADOMet)在所有细胞生物的新陈代谢中起着至关重要的作用，具有疾病中常见的改变功能。ADOMet代谢酶是化疗药物开发的靶点。本研究的目的是阐明S-腺苷甲硫氨酸合成酶(ATP：L-蛋氨酸S-腺苷转移酶，MAT)和ADOMet脱羧酶(ADOMetDC)的功能，该酶的反应使ADOMet参与多胺合成。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，并导致新的抑制剂的发现。 MAT的机理将通过计算方法来表征，以阐明其基础 这两步反应的催化作用。实验数据指导量子力学在物理中的应用 活性中心残基作用的研究。机械的推论将在实验中得到检验。通过将小分子库与MAT晶体结构进行虚拟对接，将发现新型MAT抑制剂。将对候选抑制剂进行实验评估，以确定能够调节细胞内ADOMet水平的化合物。人类病原体化脓性链球菌既有一种典型的只在古生菌中发现的垫子，也有细菌的形式。古生菌类型的垫子被假设为使用除了ATP之外的底物来合成新的代谢物；这可能提供一个新的抗生素靶点。这两个克隆的化脓性链球菌MAT都将在大肠杆菌中表达、纯化和鉴定。如果特异性假说在体外得到证实，化脓性链球菌的提取物将揭示这些新的代谢物是否在体内形成。对比了两种含有ADOMetDC的非同源丙酮酰辅因子的催化机理。动力学和结构研究将阐明激活剂非依赖类的代表和金属离子依赖基团的代表的机制。蛋白质(和金属离子)对辅因子和酶底物络合物电子环境的影响将通过选择性浓缩蛋白质和底物络合物的13C和15N核磁共振来揭示。这些研究将揭示为什么自然界保守地使用丙酮酰辅助因子来实现这种代谢功能，而不是采用常见的吡哆醛辅助因子。

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.