Investigation of Homocystinuria-Associated Mutations in the Active Site of Cystathionine β-Synthase

Abstract

Homocystinuria is an inherited autosomal recessive disorder that affects an individual's ability to metabolize the L-methionine derivative L-homocysteine. Cystathionine β-synthase (CBS) catalyzes the condensation of L-homocysteine and Lserine in the first step of the reverse transsulfuration pathway, which shunts excess Lhomocysteine to L-cysteine biosynthesis. More than 100 mutations of the CBS gene have been associated with homocystinuria. Treatment for this disease includes supplementation with vitamin B 6 and/or betaine and restriction of dietary L-methionine. Refinement of these therapeutic regimes will be enabled by an enhanced understanding of the distinct effects of homocystinuria-associated mutations as well as detailed characterization of the roles of the residues and hydrogen bonding networks in the enzyme active site. This study: (1) probed the role of active site residues D319, S323 and Y324 in the positioning of the pyridoxal 5'-phosphate cofactor of the model yeast CBS enzyme and (2) investigated a set of 11 homocystinuria-associated mutations in the context of human CBS. In the first study, substitution of D319 with alanine, removing only the carboxylate moiety of the side chain, eliminated activity, while the S323A and Y324F variants, each lacking the side chain hydroxyl group, had negligible effect on the kinetic parameters, compared to the wild-type enzyme. Residue G148 provides the mobile loop the flexibility to transition between the open and closed active site conformations. Residues E176, K384 and G307 are involved in maintaining a catalytically productive active site architecture. Residues T257 and G259 serve critical roles in PLP binding and in the bi-directional communication between the heme and PLP iii cofactors. Residues G305 and S349 also participate in positioning of the PLP cofactor.Understanding the structure-function relationships that underlie the molecular basis for each distinct homocystinuria-associated mutation of CBS provides insight that can be used to refine treatment for patients with homocystinuria. iv ACKNOWLEDGEMENTS Dr. Martin Luther King Jr. once said, "The ultimate measure of a man is not where he stands in moments of comfort and convenience, but where he stands at times of challenge and controversy". The challenge of completing my M.Sc. degree would not have been possible without the rays of light that have guided me through this tunnel.Firstly, I would like to thank Dr. Susan M. Aitken for giving me the opportunity to work in her lab as an undergraduate albeit my grades signified that I did not deserve nor earned that opportunity. Yet, you helped me realized that research is my passion. Without your insight, I would not be in the position that I am in right now.