Mechanism of the B6 Enzyme 0-Acetylserine Sulfhydrylase

OASS是一种依赖plp的酶，催化l-半胱氨酸生物合成的最后一步，即o -乙酰- l-丝氨酸和硫转化为l-半胱氨酸和乙酸。OASS是为数不多的由plp依赖性酶催化的取代和消除反应之一，已被详细研究。它是一个独特的-取代反应，具有乒乓机制，允许研究反应的两半分开。在过去的资助期内，研究主要集中在OASS的A和B同工酶的动力学和酸碱化学机制，以及中间体、结晶和定点变质的光谱鉴定。因此，动力学和酸碱化学机制是众所周知的，以及沿着反应途径的许多中间体的光谱性质。此外，OASS-A已被结晶，制备了脱酶，并研究了几个位点定向突变体。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OASS的机制，但尚不清楚沿着反应途径建立的中间体，或者决定速率的过渡态结构的性质。将使用天然底物和慢速替代底物测量同工酶的原生底物氘、溶剂氘、次生氘和多种同位素效应。2)同位素效应研究将由两个半反应的快速扫描停流研究补充。目标1和目标2应该允许对反应途径进行重构，并提供过渡态结构信息。3)许多位点导向突变体已经被用来探测与机制有关的酶残基的功能，包括K42（希夫碱赖氨酸）、C43（已知唯一的半胱氨酸，位于活性位点）和W51/W162（仅有的两个Trps之一涉及荧光e能量转移），这些突变体正在被表征。根据酶晶体结构的要求，将制备额外的位点定向突变体，这将在上述研究概述之外得到解决，但不属于本提案的一部分。这些研究的完成将使OASS成为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反应的色氨酸合成酶2进行比较。o -乙酰丝氨酸巯基水解酶是一种依赖于维生素B6的活性的酶。拟议研究的目的是确定酶及其B6辅因子如何催化氨基酸中一个基团取代另一个基团，氨基酸是蛋白质的组成部分。酶的研究是一类酶的代表，因此学到的任何东西都可以应用到课堂上的其他酶。该反应将使用动力学技术进行研究，该技术观察酶的催化作用，以及位点定向诱变，该技术允许改变酶的特定氨基酸残基，这些氨基酸残基被认为在反应中很重要。＊＊＊

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. ***