Mechanism of PLP-Dependent beta-Eliminases

我们研究的长期目标是阐明依赖于吡哆醛5‘-磷酸(PLP)的酶在氨基酸的β-碳处催化化学的机制。研究的重点是O-乙酰丝氨酸硫水解酶催化O-乙酰基-L-丝氨酸的乙酰氧基被SH取代得到L-半胱氨酸的机理。有趣的是，OASS的一部分存在于半胱氨酸合成酶(CS)与半胱氨酸生物合成途径中的第一个酶-丝氨酸乙酰转移酶(SAT)的多酶复合体中。因此，正在对单独的和CS多酶复合体中的组成酶进行完整的机理研究。在这方面，根据以下特定目的，计划进行机制研究以阐明丝氨酸乙酰转移酶的动力学和化学机制。1)通过动力学参数的pH依赖性和同位素效应来确定STA的动力学和酸碱化学机理。2)L-半胱氨酸调节SAT的机制将利用初始速度研究、同位素效应、光谱研究和分析性超速离心--依赖于PLP的酶催化的β-置换反应，因为它们产生一种β-氨基丙烯酸酯外部乙二胺中间体，一种热的电泳体。此外，这类酶有可能催化β消除和β加成反应，在协调的，E2，或逐步的，E1反应中。人们感兴趣的是确定OASS如何稳定其β-氨基丙烯酸酯中间体，以及消除和加成机制是协同的还是逐步的，即在反应的两个半过程中是否产生了苯醌中间体。此外，该酶在整个反应中的催化优势具有重要意义。最后，最近发现了一个与小阴离子结合的新的变构调节位点，并提出了一个必须进行测试的分子抑制机制。上述问题将通过以下具体目标得到回答。1)将利用稳态态和稳态态动力学技术以及动力学同位素效应来探讨反应机理，以获得关于反应后半部分的信息。2)寡核苷酸定向突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)新的调控机制将通过对变构效应传递途径上的残基进行寡核苷酸定向突变来测试。

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.