title: Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine beta-synthase.
abstract: Cystathionine beta-synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal 5'-phosphate-dependent condensation of <span class="smallcap">l</span>-serine and <span class="smallcap">l</span>-homocysteine to form <span class="smallcap">l</span>-cystathionine (<span class="smallcap">l</span>-Cth). A model of the <span class="smallcap">l</span>-Cth complex of the truncated form of yeast CBS (ytCBS), comprising the catalytic core, was constructed to identify residues involved in the binding of <span class="smallcap">l</span>-homocysteine and the distal portion of <span class="smallcap">l</span>-Cth. Residue K112 was selected for site-directed mutagenesis based on the results of the in silico docking of <span class="smallcap">l</span>-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 beta-replacement reaction is G247S asymptotically equal to K112Q > K112L asymptotically equal to K112R >> Y248F > D249A asymptotically equal to H138F > E136A. The beta-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The Km<span class="smallcap">l</span>-Ser of the K112L and K112R mutants is increased by 50- and 90-fold, respectively, while Km<span class="smallcap">l</span>-Hcys increases by only 2- and 4-fold, respectively. The Km<span class="smallcap">l</span>-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 <span class="smallcap">l</span>-Hcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation.
authors.affiliation: Department of Biology, Carleton University, Ottawa, ON, Canada.
authors.forename: Pratik H
authors.initials: PH
authors.lastname: Lodha
authors.forename: Hooman
authors.initials: H
authors.lastname: Shadnia
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Woodhouse
authors.forename: James S
authors.initials: JS
authors.lastname: Wright
authors.forename: Susan M
authors.initials: SM
authors.lastname: Aitken
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D020134:Catalytic Domain; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D003541:Cystathionine beta-Synthase