Allosteric regulation of human cystathionine beta-synthase

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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.