Mechanism of the B6 Enzyme 0-Acetylserine Sulfhydrylase

B6 酶 0-乙酰丝氨酸硫酸化酶的机制

• 批准号: 9405020
• 负责人: Paul Cook
• 金额: $ 19万
• 依托单位: University of North Texas Health Science Center at Fort Worth
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-15 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405020&HistoricalAwards=false
• 关键词: Mechanism; B6; Enzyme; Acetylserine; Sulfhydrylase

9405020 Cook O-Acetylserine sulfhydrylase (OASS) is a PLP-dependent enzyme that catalyzes the last step in the biosynthesis of L-cysteine, the conversion of O-acetyl-L-serine and sulfide to L-cysteine and acetate. he OASS is one of only a few -substitution and - elimination reactions catalyzed by PLP-dependent enzymes that has been studied in detail. It is unique as a -substitution reaction in having a ping pong mechanism, allowing study of the two halves of the reaction separately. Over the past grant period studies have focused on the kinetic and acid-base chemical mechanisms of the A and B isozymes of OASS, as well as the spectral identification of intermediates, crystallization, and site-directed metagenesis. As a result, the kinetic and acid-base chemical mechanisms are well known, as is the spectral properties of many of the intermediates along the reaction pathway. In addition, OASS-A has been crystallized, apoenzyme prepared, and several site- directed mutants have been studied. Continuation of the studies of OASS will be carried out using four main aims. 1) Although a mechanism has been proposed for OASS, it is not known what intermediates build up along the reaction pathway, or the nature of the rate-determining transition state structures. Primary substrate deuterium solvent deuterium, secondary deuterium, and multiple isotope effects will be measured for both isozymes using natural and slow alternative substrates. 2) The isotope effect studies will be complemented by rapid scanning stopped-flow studies of both half reactions. Aims 1 and 2 should allow a reconstitution of the reaction pathway and provide transition state structural information. 3) A number of site-directed mutants have ben made probing the function of enzymes residues that are implicated in aspects of mechanism including K42 (Schiff base lysine), C43 (the only cysteine and known to be in the active site), and W51/W162 (one of the only two Trps is implicated fluorescence e nergy transfer) and these are being characterized. Additional site- directed mutants will be prepared as dictated by the enzyme crystal structure, which will be solved in addition to the studies outlines above, but is not part of the present proposal. the completion of these studies will be placed OASS among the best studies of the PLP enzymes. In addition, the structure of OASS-A will be available to compare to the tryptophan synthase 2 which catalyzes a very similar reaction. %%% O-Acetylserine sulfhydrylase is an eznyme that depends on a form of vitamin B6 for its activity. The purpose of the proposed studies is to determined how the enzyme and its B6 cofactor catalyze the substitution of one group for another in amino acids, the building block of proteins. The enzyme studies is representative of a class of enzymes, and thus anything learned could be applied to other enzymes in the class. The reaction will be studied using kinetic techniques which view the enzyme in the act of catalysis, and site directed mutagenesis which allows a change in specific amino acid residues of the enzyme thought to be important in the reaction. ***

9405020 Cook O-乙酰丝氨酸硫化氢解酶（OASS） 是一种PLP依赖性酶，催化L-半胱氨酸生物合成的最后一步，O-乙酰基-L-丝氨酸和硫化物转化为L-半胱氨酸和乙酸盐。 OASS是少数几个已被详细研究的由PLP依赖性酶催化的取代和消除反应之一。 它是一个独特的-取代反应，在有一个乒乓机制，允许研究的两个半的反应分别。 在过去的资助期间，研究集中在动力学和酸碱化学机制的A和B同工酶的OASS，以及光谱鉴定的中间体，结晶和定点后生。 因此，动力学和酸碱化学机制是众所周知的，因为是许多中间体的光谱特性沿着反应途径。 此外，OASS-A已结晶，脱辅基酶制备，并已研究了几个定点突变体。 将利用四个主要目标继续进行OASS的研究。 1)虽然已经提出了一种机制OASS，它是不知道什么中间体建立了沿着反应途径，或性质的速率决定过渡态结构。 主要底物氘溶剂氘，二级氘，和多种同位素效应将使用天然和缓慢的替代底物的两种同工酶进行测量。 2)同位素效应研究将通过两个半反应的快速扫描停流研究来补充。目标1和2应允许反应途径的重构并提供过渡态结构信息。 3)许多定点突变体已经被制成探测涉及机制方面的酶残基的功能，包括K42（席夫碱赖氨酸）、C43（已知在活性位点中的唯一半胱氨酸）和W51/W162（仅有的两个Trps之一涉及荧光能量转移），并且这些突变体正在被表征。 根据酶晶体结构的要求，将制备其他定点突变体，除了上述研究外，还将解决这些问题，但这些问题不是本提案的一部分。 这些研究的完成将被置于 OASS是PLP酶的最佳研究之一。 此外，OASS-A的结构将可用于与催化非常相似的反应的色氨酸合酶2进行比较。 O-Acetylserine sulfhydrase是一种酶，它依赖于维生素B6的一种形式来发挥其活性。 拟议研究的目的是确定酶及其B6辅因子如何催化氨基酸（蛋白质的构建单元）中一个基团取代另一个基团。 酶研究代表了一类酶，因此所学到的任何知识都可以应用于该类别中的其他酶。 将使用动力学技术研究反应，动力学技术观察催化作用中的酶，以及定点诱变，定点诱变允许改变被认为在反应中重要的酶的特定氨基酸残基。 ***