Allosteric regulation of human cystathionine beta-synthase

人胱硫醚β-合酶的变构调节

• 批准号: 10602404
• 负责人: Joseph V. Roman
• 金额: $ 6.95万
• 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/10602404
• 关键词: Active Sites; Address; Affect; Allosteric Regulation; Anabolism; 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; Dimerization; 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; Oxidation-Reduction; Pathogenicity; Pathway interactions; Patients; Physical condensation; Process; Proteins; Pyridoxal Phosphate; Reaction; Recombinants; Regulation; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Serine; Site; Skeletal system; Spectrophotometry; Spectrum Analysis; Structure; Sulfur; Testing; Translating; Variant; Visual System; Water; X-Ray Crystallography; body system; cofactor; conformational conversion; 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.

项目摘要 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）。该途径中的第一种酶，胱硫醚β- 合成酶（CBS）导致高胱氨酸尿症并影响四个主要器官系统。CBS催化缩合 丝氨酸和同型半胱氨酸的结合，产生胱硫醚和水。或者，它可以催化缩合 半胱氨酸和同型半胱氨酸的结合，产生胱硫醚和硫化氢。CBS是一种模块化蛋白质，其中中央 催化结构域的两侧是N-末端血红素结构域和C-末端S-腺苷甲硫氨酸（S-Met） 这两个领域都是监管领域。长距离通讯参与CBS的变构调节， 血红素和活性位点之间的距离为~20 μ m，血红素和蛋氨酸位点之间的距离为~50 μ m - 是的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，并且是 预计会干扰由血红素甲硫氨酸引起的变构调节，进而干扰血红素甲硫氨酸反应性调节 域我推测，接头突变不利于从基础到基础的构象转变。 激活状态，该状态是由触发器触发的。我将通过以下目标来检验我的假设。(i)我会 表征致病性接头突变（G347 S、K384 E/N和M39 I）的稳态动力学参数 在典型的和H2S产生的反应催化的CBS和结合常数的蛋氨酸。我会 评估接头突变对硫通过转硫途径的通量的影响。(ii)我会 通过测定血红素的还原，研究连接子突变对血红素氧化还原环境的影响。 结合血红素的潜力。CO和NO·与亚铁血红素结合的动力学和结合常数 将通过停流分光光度法测定是否存在蛋氨酸。(iii)我会把 接头突变体，并确定全长CBS的结构。成功完成这些研究将 拓宽我们对CBS如何监管的理解，并加深对远程监管机制的理解。 远端调控域之间的通信。