Mechanism of the B6 Enzyme 0-Acetylserine Sulfhydrylase

9405020库克O-乙酰丝氨酸硫基酶是一种依赖磷脂酶原激活剂的酶，催化L-半胱氨酸生物合成的最后一步，即O-乙酰基-L-丝氨酸和硫化物转化为L-半胱氨酸和乙酸酯。OASS是少数几个被详细研究过的PLP依赖酶催化的取代和消除反应之一。它作为-取代反应的独特之处在于具有乒乓球机制，允许分别研究反应的两个半部分。在过去的赠款期间，研究的重点是OASS A和B同工酶的动力学和酸碱化学机制，以及中间体、结晶和定点化生的光谱识别。因此，动力学和酸碱化学机制是众所周知的，反应路径上的许多中间体的光谱性质也是众所周知的。此外，还对OASS-A进行了结晶，制备了脱辅酶，并研究了几种定点突变体。继续开展对东非国家组织的研究将有四个主要目标。1)虽然已经提出了OASS的机理，但还不知道中间体在反应路径上积累了什么，也不知道决定速度的过渡态结构的性质。使用自然底物和慢速替代底物测量两种同工酶的初级底物、溶剂重氢、次要重氢和多同位素效应。2)同位素效应研究将得到两个半反应的快速扫描停流研究的补充。AIMS 1和AIMS 2应允许重新构建反应路径，并提供过渡态结构信息。3)一些定点突变体已经探索了与机制有关的酶残基的功能，包括K42(Schiff碱基赖氨酸)、C43(已知的唯一位于活性部位的半胱氨酸)和W51/W162(仅有的两个TRPs中的一个与荧光能量转移有关)，这些突变正在被鉴定。将根据酶的晶体结构制备更多的定点突变体，这将在上面的研究大纲之外得到解决，但不是本提案的一部分。这些研究的完成将使OASS跻身于PLP酶的最佳研究之列。此外，OASS-A的结构将可用于与色氨酸合成酶2进行比较，色氨酸合成酶2催化的反应非常相似。%O-乙酰丝氨酸巯基酶是一种依赖于维生素B6形式的酶。这项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的一个基团替代另一个基团，氨基酸是蛋白质的组成部分。酶的研究代表了一类酶，因此所学的任何东西都可以应用于这一类的其他酶。将使用动力学技术来研究该反应，该技术将酶视为催化作用，以及定点突变，它允许改变被认为在反应中重要的酶的特定氨基酸残基。***

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. ***