Mechanism of the B6 Enzyme O-Acetylserine Sulfhydrylase

这项工作将进一步加深我们对o -乙酰丝氨酸巯基化酶特异性和一般PLP酶催化的-取代反应机理的理解。两个具体目的：首先，阐明酶催化的酸碱化学。这方面将利用初始速度研究来测量与底物竞争的抑制剂的各种值作为ph的函数。此外，沿着反应途径的具有有限寿命的光谱中间体将被滴定，以确定酶和/或辅因子的pK值是否随着反应进行而受到干扰。其次，确定汇率确定步骤限制的位置和数量。这些研究将利用原生和次生氘、溶剂氘和多同位素效应及其pH依赖性。同位素效应数据应该允许精确定位速率决定步骤的位置，并允许解释共价催化的机制框架。最后，用180标记酯羰基氧的位置同位素交换来确定前半反应中的产物释放是部分限速还是完全限速。这里提出的广泛的实验可能会给这种重要的酶的作用模式提供相当详细的见解，而这些信息可能会在以后的一些关于蛋白质-蛋白质相互作用的催化后果的有趣研究中使用，这一前景为该项目提供了一个额外的维度。库克博士完全有资格从事这项工作。

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.