Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine β-synthase
Cystathionine β-synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal 5′-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine (L-Cth). A model of the L-Cth complex of the truncated form of yeast CBS (ytCBS), comprising the catalytic core, was constructed to identify residues involved in the binding of L-homocysteine and the distal portion of L-Cth. Residue K112 was selected for site-directed mutagenesis based on the results of the in silico docking of L-Cth to the modeled structure of ytCBS. Residues E136, H138, Y248, and D249 of ytCBS were also targeted as they correspond to identical polar residues lining the mouth of the active site in the structure of human CBS. A series of 8 site-directed mutants was constructed, and their order of impact on the ability of ytCBS to catalyze the β-replacement reaction is G247S ≈ K112Q > K112L ≈ K112R ≫ Y248F > D249A ≈ H138F > E136A. The β-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The Km L-Ser of the K112L and K112R mutants is increased by 50- and 90-fold, respectively, while Km L-Hcys increases by only 2- and 4-fold, respectively. The Km L-Hcys of H138F and Y248F is increased by 8- and 18-fold, respectively. These results indicate that, while the targeted residues are not direct determinants of L-Hcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation.
|Keywords||Cystathionine, Homocysteine, Homocystinuria, Pyridoxal 5′-phosphate, Transsulfuration|
|Journal||Biochemistry and Cell Biology|
Lodha, P.H. (Pratik H.), Shadnia, H. (Hooman), Woodhouse, C.M. (Colleen M.), Wright, J.S, & Aitken, S. (2009). Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine β-synthase. Biochemistry and Cell Biology, 87(3), 531–540. doi:10.1139/O09-003