Allosteric regulation of human cystathionine beta-synthase

项目摘要 硫磺代谢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(硫化氢)。该途径中的第一种酶--胱硫醚β--的功能障碍 合成酶(CBS)导致同型半胱氨酸尿症，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，生成胱硫氨酸和水。或者，它可以催化缩合。 半胱氨酸和同型半胱氨酸，生成胱硫氨酸和硫化氢。CBS是一种模块化蛋白质，在这种蛋白质中， 催化结构域的侧翼是一个N-末端的血红素结构域和一个C-末端的S腺苷蛋氨酸(ADOMet) 这两个领域都是受监管的。CBS的变构调节与远程通讯有关 亚铁血红素与活性中心之间的距离约为20？，亚铁血红素与阿多梅特中心之间的距离约为50 那就是。患者突变的一个子集映射到催化和C末端结构域之间的连接区，并且是 预计会扰乱ADOMet的变构调节，进而扰乱ADOMet对血红素的响应调节 域。我假设连接子的突变不利于从基端到基端的构象转变 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 表征致病链接子突变(G347S、K384E/N和M39I)的稳态动力学参数 在CBS催化的正则反应和生成H_2S的反应中，计算了ADO-Met的结合常数。这就做 评估连接子突变对通过硫磺转化途径的硫磺通量的影响。(Ii)我会 通过测定还原来研究连接子突变对血红素氧化还原环境的影响 结合的亚铁血红素的势。CO和NO·与亚铁血红素结合的动力学常数和结合常数 ADOMet的存在和不存在将通过停流分光光度法来确定。(Iii)我会明确 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监管方式的理解，深化对长效机制的洞察 远端调控结构域之间的通信。

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.