ENZYME CATALYZED BETA ELIMINATION

这项研究的总体目标是阐明催化 酶催化PLP依赖性β-消除的机制 反应 将进行研究以确定动力学机制， 速率限制步骤沿着反应坐标的位置， 化学反应发生在底物周转过程中的活性位点， 酵素 可用于获取这些信息的实验技术 包括在不存在和存在产品的情况下的初始速率研究，或 死端抑制剂、pH和同位素效应研究。 主要目的是 反应配位与速率常数的描述 步骤，并阐明过渡态和任何 中间体的 关于化学机理的信息可以从 动力学参数（V，V/K和Ki）的pH依赖性， 抑制剂和底物），以及来自初级和次级同位素效应。 这项建议可分为四个部分。 第一，确定 对E.大肠杆菌色氨酸吲哚 裂合酶 与平衡常数有关的适当Haldle方程 （将独立测量）与动力学参数的关系将是 用于检查机制的内部一致性。 此外，pH和 用这种酶的缓慢交替底物进行的同位素效应研究将 进行。 第二，测量次级氘同位素 色氨酸吲哚裂解酶的影响，以及初级和次级 色氨酸吲哚裂解酶和酪氨酸 C. freundii和E.具有S-甲基-L半胱氨酸的西葫芦， 并在D2 O中测定这些效应。 第三，确定 两种酪氨酸酚裂解酶的溶剂氘同位素效应， 色氨酸吲哚裂解酶的反向。 质子库存将 确定上述酶。 第四，动力学测量 参数和主要氘同位素效应和pH值研究， 确定PLP依赖酶的催化机制，O- 磷酸乙醇胺磷酸裂解酶。 实际上没有机械的 关于这种酶的数据，它催化O- 磷酸乙醇胺（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有助于我们进一步研究PEA磷酸裂解酶。 磷酰基转移机制的整体理解，因为这种酶 这一机制在这方面似乎是独一无二的。 此外，这项酶的研究 催化相同类型反应的系统将提供第一个- 通过系统使用三种催化机制的深入比较 多种同位素效应（初级和次级氘和溶剂 氘）。 从研究中获得的知识应该扩大我们的 理解和解释溶剂氘同位素效应。

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.