Mechanism of PLP-Dependent beta-Eliminases

PLP 依赖性 β-消除酶的机制

• 批准号: 0111024
• 负责人: Paul Cook
• 金额: $ 9万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0111024&HistoricalAwards=false
• 关键词: Mechanism; PLP; Dependent; beta; Eliminases

The long-term goal of our studies is to elucidate the mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes that catalyze chemistry at the beta-carbon of amino acid. Studies have focused on the mechanism of the enzyme O-acetylserine sulfhydryase (OASS), which catalyzes the beta-replacement of the acetoxy group of O-acetyl-L-serine with SH to give L-cysteine. Of interest, a fraction of the OASS is in the cysteine synthetase (CS) multienzyme complex with the first enzyme in the cysteine biosynthetic pathway, serine acetyltransferase(SAT). Thus, a complete mechanistic study of the component enzymes alone and in the CS multienzyme complex is being carried out. In this regard, mechanistic studies are planned to elucidate the kinetic and chemical mechanisms of serine acetyltransferase according to the following specific aims. 1) The kinetic and acid-base chemical mechanism of STA will be determined via the pH dependence of kinetic parameters, and isotope effects. 2) The mechanismof SAT regulation by L-cysteine will make use of initial velocity studies, isotope effects, spectral studies, and analytical ultracentrifugation beta-Replacement reactions catalyzed by PLP-dependent enzymes are of interest since they generate an beta-aminoacrylate external aldimine intermediate, a hot electrophile. In addition, there is the possibility that this class of enzymes catalyze the beta elimination and beta addition reactions in a concerted, E2 , or stepwise, E1 , reaction. It is of interest to determine how OASS stabilizes its beta-aminoacrylate intermediate, and whether the mechanisms of elimination and addition are concerted or stepwise, that is whether a quinonoid intermediate is generated during the two halves of the reaction. In addition, the participation of the enzyme in the overall reaction in terms of catalytic advantage is of import. Finally, a new allosteric regulatory site for the binding of small anions has recently been identified, and a molecular mechanism of inhibition proposed that must be tested. The above questions will be answered via the following specific aims. 1) The reaction mechanism will be probed using presteady state and steady state kinetic techniques, and kinetic isotope effects to obtain information on the second half of the reaction. 2) Oligonucleotide-directed mutagenesis will be used to identify the function of enzyme residues that interact with the cofactor, and substrate. 3) The new mechanism of regulation will be tested using oligonucleotide-directed mutagenesis of residues along the pathway for transmission of the allosteric effect.

本研究的长期目标是阐明吡哆醛5 '-磷酸（PLP）依赖性酶催化氨基酸β-碳化学的机制。研究集中于酶O-乙酰丝氨酸巯基解酶（OASS）的机制，其催化O-乙酰-L-丝氨酸的乙酰氧基被SH β-取代以产生L-半胱氨酸。 感兴趣的是，OASS的一部分在半胱氨酸合成酶（CS）多酶复合物中，其中第一种酶是半胱氨酸生物合成途径中的丝氨酸乙酰转移酶（SAT）。因此，一个完整的机制研究的组分酶单独和CS多酶复合物正在进行。在这方面，机制研究计划阐明丝氨酸乙酰转移酶的动力学和化学机制，根据以下具体目标。1)STA的动力学和酸碱化学机制将通过动力学参数的pH依赖性和同位素效应来确定。2)SAT regulation的L-半胱氨酸的mechanismof将利用初速度的研究，同位素效应，光谱研究，和分析ultracentrationbeta-Replacement反应催化的PLP依赖性酶的兴趣，因为它们产生的β-氨基丙烯酸酯外部醛亚胺中间体，热亲电试剂。此外，这类酶有可能在协同E2或逐步E1反应中催化β消除和β加成反应。有趣的是，确定OASS如何稳定其β-氨基丙烯酸酯中间体，以及消除和加成的机制是协同的还是逐步的，即在反应的两个半部分期间是否生成喹喔啉中间体。此外，酶在整个反应中的催化优势是重要的。最后，一个新的小阴离子的结合变构调节位点最近已被确定，和抑制的分子机制提出，必须进行测试。上述问题将通过以下具体目标来回答。1)反应机理将使用presteady状态和稳态动力学技术，和动力学同位素效应，以获得信息的反应的下半部分进行探讨。2)寡核苷酸定向诱变将用于鉴定与辅因子和底物相互作用的酶残基的功能。3)新的调节机制将使用沿着变构效应传递途径的残基的沿着的阿糖胞苷定向诱变来测试。