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; 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 调节过硫途径是维持健康水平的硫代谢物的关键， 同型半胱氨酸和硫化氢（H2S）。在该途径中，第一种酶的功能障碍，胱淀粉β- 合酶（CBS）会导致同卵巢尿症，并影响四个主要器官系统。 CBS催化冷凝 丝氨酸和同型半胱氨酸，产生胱淀粉和水。或者，它可以催化冷凝水 半胱氨酸和同型半胱氨酸，产生胱淀粉和H2。 CBS是一种模块化蛋白 催化结构域是N末端血红素结构域和C末端S-腺苷甲氨酸（ADOMET）的侧面 域，两者都是调节性的。远程沟通与CB的变构调节有关 血红素和活性位点之间的距离约为20Å，血红素和ADOMET位置之间的距离为〜50 一个。患者突变的子集映射到催化和C末端结构域之间的接头区域，IS 预测通过ADOMEM和ADOMET响应性调节血红素的扰动变构调节 领域。我假设接头突变不利于从基本到基础的构象转变 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）我会的 表征致病连接器突变（G347S，K384E/N和M39I）的稳态动力学参数 在CBS催化的规范和H2S产生反应中，ADOMET的结合常数。我会 评估接头突变对硫通过过渡途径的通量的影响。 （ii）我会的 通过确定降低来研究接头突变对血红素氧化还原环境的影响 结合血红素的潜力。 CO和NO的动力学和结合常数•与铁血红素结合 ADOMET的存在和不存在将由停止流量分光光度法确定。 （iii）我将结晶 接头突变体并确定全长CBS的结构。这些研究的成功完成将 扩大我们对CBS如何调节的理解，并加深对远程机制的见解 远端监管域之间的通信。