STUDIES OF ENZYME CATALYZED BETA-ELIMINATION

这项研究的总体目标是阐明催化 酶催化PLP依赖的β-消除作用机制(S) 反应。将进行研究以确定动力学机制， 限速步骤沿反应坐标的位置，以及 在底物周转期间在活性部位发生的化学作用 酵素。可用于获取这些信息的实验技术 包括在没有和存在产品或 死胡同抑制剂，pH和同位素效应研究。主要目的是 用个体反应速率常数描述反应配位 步骤，并阐明过渡态和任何 中间体。有关化学机理的信息可以从 动力学参数(V、V/K和KI)与pH的关系 抑制剂和底物)，以及初级和次级同位素效应。 这项提议可以分为四个部分。第一，确定 大肠杆菌色氨酸吲哚逆转反应的动力学机理-- 裂解酶。与平衡常数相关的适当的霍尔丹方程 (将独立测量)到动力学参数 用于检查机构的内部一致性。此外，酸碱度和 这种酶的缓慢交替底物的同位素效应研究将 要承担的责任。第二，二次氚同位素的测量 色氨酸吲哚裂解酶的作用，以及原发和继发 色氨酸吲哚裂解酶和酪氨酸的氚同位素效应 弗氏拟青霉和草本拟青霉的苯酚裂解酶与S-甲基-L半胱氨酸， 以及D2O中这些影响的测定。第三，确定 酪氨酸苯酚裂解酶和In的溶剂氢同位素效应 色氨酸吲哚裂解酶的相反方向。质子库存将 对上述酶进行检测。第四，动力学的测量 参数和初级氚同位素效应和pH研究 确定PLP依赖酶O-的催化机理 磷酸乙醇胺磷酸裂解酶。实际上没有机械性的 该酶催化依赖PLP的O-水解酶的数据 将磷乙醇胺(PEA)转化为乙醛、氨和正磷酸盐。 对PEA磷酸裂解酶催化机理的了解将进一步推动我们的工作 对这种酶的磷酰转移机制有全面的认识 在这方面，该机制似乎是独一无二的。另外，这项关于酶的研究 催化相同类型的反应的系统将提供第一个in-in- 三种催化机理的系统应用深度比较 多种同位素效应(初级和次级氢和溶剂 重氢)。这项研究所获得的知识应该会拓宽我们的 理解和解释溶剂中的氚同位素效应。

The overall goal of this research is to elucidate the catalytic mechanism(s) of enzymes catalyzing a PLP-dependent beta-elimination reaction. Studies will be carried out to determine the kinetic mechanism, location of rate-limiting steps along the reaction coordinate, and the chemistry taking place during substrate turnover in the active site of the enzyme. Experimental techniques available to acquire this information include initial rate studies in the absence and presence of products or dead-end inhibitors, pH and isotope effect studies. The main purpose is delineation of the reaction coordination with rate constants for individual steps, and elucidation of the structure of the transition-state and of any intermediates. Information on the chemical mechanism can be deduced from the pH dependence of the kinetic parameters (V, V/K, and the Ki for inhibitors and substrates), and from primary and secondary isotope effects. This proposal may be divided into four parts. First, determination of the kinetic mechanism of the reverse reaction for E. coli tryptophan indole- lyase. The appropriate Haldane equations relating the equilibrium constant (which will be measured independently) to the kinetic parameters will be utilized to check for internal consistency of the mechanism. Also, pH and isotope effect studies with slow alternate substrates for this enzyme will be undertaken. Second, measurement of the secondary deuterium isotope effects for tryptophan indole-lyase, and the primary and secondary deuterium isotope effect for tryptophan indole-lyase and both tyrosine phenol-lyases from C. freundii and E. herbicola with S-methyl-L cysteine, and determination of these effects in D2O. Third, determination of the solvent deuterium isotope effects for both tyrosine phenol-lyases, and in the reverse direction for tryptophan indole-lyase. Proton inventories will be determined for the above enzymes. Fourth, measurement of the kinetic parameters and primary deuterium isotope effects and pH studies to determine the catalytic mechanism of the PLP-dependent enzyme, O- phosphorylethanolamine phospho-lyase. There is virtually no mechanistic data on this enzyme which catalyzes the PLP-dependent hydrolysis of O- phosphorylethanolamine (PEA) to acetaldehyde, ammonia, and orthophosphate. Knowledge of he catalytic mechanism for PEA phospho-lyase will further our overall understanding of phosphoryl transfer mechanisms as this enzymes mechanism appears to be unique in that regard. Also, this study of enzyme systems catalyzing the same type of reaction will provide the first in- depth comparison of three catalytic mechanisms through the systematic use of multiple isotope effects (primary and secondary deuterium and solvent deuterium). The knowledge derived for the study should broaden our understanding and interpret solvent deuterium isotope effects.