Mechanism of the B6 Enzyme O-Acetylserine Sulfhydrylase

这项工作的开展将进一步加深我们对 O-乙酰丝氨酸硫化氢解酶的作用机制， - 通常由PLP酶催化的置换反应。 两个具体目标：第一， 酶将被分解。 这方面将利用 初始速度研究，以测量 与底物竞争的抑制剂作为pH的函数。 此外，光谱中间体沿着反应途径 将滴定有限的寿命，以确定是否 酶和/或辅因子pK值随着反应的进行而受到干扰 收益。 第二，限制的位置和数额 将确定速率确定步骤。 这些研究将 利用伯氘、仲氘、溶剂氘 以及多种同位素效应和它们的pH依赖性。 的 同位素效应数据应能确定 率确定步骤，并允许一个机械框架， 共价催化的解释。 最后，位置 同位素交换，其中酯羰基氧被标记 180将用于确定产品是否在 第一半反应被部分或完全地速率化 限制。 这里提出的广泛的实验可能会给 更详细地了解了 这种重要的酶现在已经可以使用了，前景 这些信息可能会被用于一些有趣的 蛋白质-蛋白质催化反应的研究 互动为项目提供了一个额外的维度。 博士 库克完全有资格开展这项工作。

This work will be carried out to further our understanding of the mechanism of O-acetylserine sulfhydrylase specifically and -replacement reactions catalyzed by PLP enzymes in general. Two specific aims: First, the acid-base chemistry catalyzed by enzyme will be elucidated. This aspect will make use of initial velocity studies to measure various values for inhibitors competitive with the substrates as a function of pH. In addition, spectral intermediates along the reaction pathway with a finite lifetime will be titrated to determine whether enzyme and/or cofactor pK values are perturbed as the reaction proceeds. Second, the location and amount of limitation of rate determining steps will be determined. These studies will make use of primary and secondary deuterium, solvent deuterium and multiple isotope effects and their pH dependence. The isotope effect data should allow pinpointing the location of rate determining steps and permit a mechanistic framework for interpretation of covalent catalysis. Finally, positional isotope exchange in which the ester carbonyl oxygen is labeled with 180 will be used to determine whether product release in the first half-reaction is partially or completely rate limiting. The broad range of experiments proposed here is likely to give considerably more detailed insight into the mode of action of this important enzyme then is available today, and the prospect that this information may later on be used in some intriguing studies on the catalytic consequences of protein-protein interaction gives the project an additional dimension. Dr. Cook is well qualified to carry out this work.