Allosteric regulation of human cystathionine beta-synthase

人胱硫醚β-合酶的变构调节

• 批准号: 10381000
• 负责人: Joseph V. Roman
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10381000
• 关键词: Active Sites; Address; Affect; Allosteric Regulation; Anaerobic Bacteria; Binding; Biochemical; Biochemical Pathway; Biogenesis; Biological Assay; C-terminal; Cardiovascular system; Catalytic Domain; Colon Adenocarcinoma; Communication; Complement; Couples; Crystallization; Cystathionine; Cystathionine beta-Synthase; Cysteine; Cysteine Desulfhydrase; Data; Disease; Distal; Environment; Enzymes; Equilibrium; Exhibits; Functional disorder; HT29 Cells; Heme; Hemeproteins; Homocysteine; Homocystinuria; Human; Hydrogen Sulfide; In Vitro; Inherited; Kinetics; Laboratories; Length; Mammalian Cell; Maps; Mediating; Missense Mutation; Molecular; Molecular Conformation; Mutation; N-terminal; Nervous system structure; Oxidation-Reduction; Pathogenicity; Pathway interactions; Patients; Physical condensation; Process; Proteins; Pyridoxal Phosphate; Reaction; Recombinants; Regulation; Role; S-Adenosylmethionine; Serine; Site; Skeletal system; Spectrophotometry; Spectrum Analysis; Structure; Sulfur; Testing; Translating; Variant; Visual system structure; Water; X-Ray Crystallography; body system; cofactor; conformational conversion; dimer; flexibility; heme a; insight; interest; lanthionine; monomer; mutant; patient subsets; potentiometric titration; respiratory toxin; stable cell line; tautomer

Project Summary Regulation of the transsulfuration pathway is key to maintaining healthy levels of the sulfur metabolites, homocysteine and hydrogen sulfide (H2S). Dysfunction of the first enzyme in this pathway, cystathionine β- synthase (CBS) results in homocystinuria and affects four major organ systems. CBS catalyzes the condensation of serine and homocysteine, generating cystathionine and water. Alternatively, it can catalyze the condensation of cysteine and homocysteine, generating cystathionine and H2S. CBS is a modular protein in which the central catalytic domain is flanked by an N-terminal heme domain and a C-terminal S-adenosylmethionine (AdoMet) domain, both of which are regulatory. Long range communication is involved in allosteric regulation of CBS with the distance between the heme and active site being ~20 Å and between the heme and AdoMet sites, being ~50 Å. A subset of patient mutations map to the linker region between the catalytic and C-terminal domains, and is predicted to perturb allosteric regulation by AdoMet and in turn, AdoMet-responsive regulation of the heme domain. I hypothesize that the linker mutations disfavor the conformational transition from the basal to the activated state that is triggered by AdoMet. I will test my hypothesis by addressing the following aims. (i) I will characterize the steady-state kinetic parameters of the pathogenic linker mutations (G347S, K384E/N, and M39I) in the canonical and H2S-producing reactions catalyzed by CBS and the binding constant for AdoMet. I will assess the impact of the linker mutations on the flux of sulfur through the transsulfuration pathway. (ii) I will investigate the effects of the linker mutations on the heme redox environment by determining the reduction potential of the bound heme. The kinetic and binding constants of CO and NO• binding to ferrous heme in the presence and absence of AdoMet will be determined by stopped-flow spectrophotometry. (iii) I will crystallize the linker mutants and determine the structure of full-length CBS. Successful completion of these studies will broaden our understanding of how CBS is regulated and deepen insights into the mechanism of long-range communication between distal regulatory domains.

项目概要 转硫途径的调节是维持硫代谢物健康水平的关键， 同型半胱氨酸和硫化氢（H2S）该途径中第一种酶胱硫醚β-的功能障碍。 合成酶 (CBS) 会导致同型半胱氨酸尿症并影响四个主要器官系统。 丝氨酸和同型半胱氨酸，生成胱硫醚和水，或者，它可以催化缩合。 半胱氨酸和同型半胱氨酸，生成胱硫醚和H2S，这是一种模块化蛋白质，其中的核心。 催化结构域两侧是 N 端血红素结构域和 C 端 S-腺苷甲硫氨酸 (AdoMet) 域，两者都是调节性的，涉及 CBS 的变构调节。 血红素和活性位点之间的距离约为 20 Å，血红素和 AdoMet 位点之间的距离约为 50 Å. 患者突变的子集映射到催化结构域和 C 末端结构域之间的接头区域，并且是 预计会扰乱 AdoMet 的变构调节，进而扰乱 AdoMet 血红素响应性调节 我认为接头突变不利于从基础结构域到结构域的构象转变。 由 AdoMet 触发的激活状态 我将通过解决以下目标来检验我的假设 (i) 我将。 表征致病性接头突变（G347S、K384E/N 和 M39I）的稳态动力学参数 在 CBS 催化的典型反应和 H2S 生成反应中以及 AdoMet 的结合常数。 评估接头突变对通过转硫途径的硫通量的影响。 通过确定还原来研究接头突变对血红素氧化还原环境的影响 结合血红素的势能 CO 和 NO• 与亚铁血红素结合的动力学和结合常数。 AdoMet 的存在和不存在将通过停流分光光度法测定 (iii) 我将结晶。 连接子突变体并确定全长 CBS 的结构将成功完成这些研究。 拓宽我们对CBS监管方式的认识，加深对长程机制的认识 远端调节域之间的通信。