Mechanism of the B6 Enzyme O-Acetylserine Sulfhydrylase

B6 酶 O-乙酰丝氨酸硫酸化酶的机制

• 批准号: 8812701
• 负责人: Paul Cook
• 金额: $ 6万
• 依托单位: University of North Texas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-15 至 1991-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8812701&HistoricalAwards=false
• 关键词: Mechanism; B6; Enzyme; Acetylserine; Sulfhydrylase

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.

这项工作的开展将进一步加深我们对O-乙酰丝氨酸巯基酶的特异性机理和PLP酶催化的-取代反应的一般理解。具体目标有两个：第一，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缓蚀剂的各种值作为pH的函数。此外，将滴定具有有限寿命的反应路径上的光谱中间体，以确定酶和/或辅因子的pk值是否随着反应的进行而受到干扰。其次，将确定费率确定步骤的限制位置和数量。这些研究将利用初级和次级氢、溶剂氢和多同位素效应及其与pH的关系。同位素效应数据应该能够精确地确定速率决定步骤的位置，并允许解释共价催化的机理框架。最后，位置同位素交换将被用来确定第一半反应中的产物释放是部分还是完全限速。这里提出的广泛的实验范围可能会给出比现在更详细的对这种重要酶的作用模式的洞察，并且这些信息可能在以后用于一些有趣的关于蛋白质-蛋白质相互作用的催化结果的研究的前景给该项目提供了一个额外的维度。库克博士完全有资格开展这项工作。