Mechanism of the B6 Enzyme, O-Acetylserine Sulfhydrylase

B6 酶 O-乙酰丝氨酸硫酸化酶的机制

• 批准号: 9729609
• 负责人: Paul Cook
• 金额: $ 27万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9729609&HistoricalAwards=false
• 关键词: Mechanism; B6; Enzyme; Acetylserine; Sulfhydrylase

9729609 Cook The objective of this research is a determination of the mechanism of O-acetylserine sulfhydrylase (OASS-A), a pyridoxal phosphate (PLP) dependent enzyme . The kinetic and chemical mechanisms of OASS-A have been determined, and the three dimensional structure of the enzyme has been solved. A number of questions still remain concerning the mechanism. The slow steps in the first half of the reaction have been identified, but virtually nothing is known concerning the second half of the reaction. In this project, the reaction mechanism will be probed using presteady state and steady state kinetic techniques. Kinetic isotope effects will be used to obtain information on the second half of the reaction. The stereochemistry of the elimination of acetic acid and the addition of H2S, and whether a quinonoid intermediate exists along the reaction pathway will also be examined. Oligonucleotide-directed mutagenesis will be used to identify the enzyme residue that interacts with the acetyl side chain of OAS, groups that interact with the cofactor, and residue(s) involved in binding the alpha-carboxyl of OAS. The long term goal remains an elucidation of the mechanism of the multienzyme complex cysteine synthetase, composed of serine transacetylase and OASS. A complete description of the mechanisms of the component enzymes alone and in complex will be determined. The present project, however, focuses on the study of OASS-A. A study of the mechanism catalyzed by OASS-A should significantly increase our understanding of PLP enzymes in general, and those that catalyze beta-replacement reactions. Isotope effects is a technique that allows one to look at the structure of the activated complex (transition state) as reactant is converted to product, and the types of bonds formed and broken during the OASS reaction and the differences in chemistry compared to other PLP enzymes make OASS-A of interest. A knowledge of transition state structure provides a basis f or bactericides for PLP-dependent enzymes catalyzing beta-replacement reactions. The OASS-A reaction provides potential for the production of a number of novel beta-substituted amino acids.

9729609库克这项研究的目的是确定O-乙酰丝氨酸硫基酶(OASS-A)的作用机制，该酶是一种依赖磷酸吡哆醛(PLP)的酶。确定了OASS-A的动力学和化学机理，解决了该酶的三维结构问题。关于这一机制，仍然存在一些问题。已经确定了反应前半部分的缓慢步骤，但对反应的后半部分几乎一无所知。在本项目中，将使用稳态前动力学技术和稳态动力学技术来探讨反应机理。动力学同位素效应将被用来获得关于反应后半部分的信息。还将研究消除醋酸和添加硫化氢的立体化学，以及反应路径上是否存在类醌中间体。寡核苷酸定向突变将被用于鉴定与OAS的乙酰基侧链相互作用的酶残基、与辅因子相互作用的基团以及参与结合OAS的α-羧基的残基(S)。长期的目标仍然是阐明由丝氨酸转乙酰酶和OASS组成的多酶复合半胱氨酸合成酶的机制。将确定组成酶单独的和复杂的机制的完整描述。然而，本项目侧重于对OASS-A的研究。对OASS-A催化机制的研究将大大增加我们对PLP酶的总体理解，以及那些催化β-取代反应的酶。同位素效应是一种技术，它允许人们在反应物转化为产物时观察激活的络合物(过渡态)的结构，以及在OASS反应中形成和断裂的键的类型以及与其他PLP酶相比的化学差异，从而使OASS-A引起人们的兴趣。对过渡态结构的了解为PLP依赖的酶催化β-取代反应提供了杀菌剂的基础。OASS-A反应为生产许多新型的β-取代氨基酸提供了可能性。