Summary
Publication: Biotech Week
“Cystathionine beta-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,” scientists in Ottawa, Canada report (see also ).
“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 beta-replacement reaction is G247S approximate to K112Q > K112L approximate to K112R >> Y248F > D249A approximate to H138F > E136A. The beta-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The K-m(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 K-m(L-Hcys) of H138F and Y248F is increased by 8- and 18- fold, respectively,” wrote P.H. Lodha and colleagues, Carleton University.
The researchers concluded: “These results indicate that, while the targeted residues are not direct determinants of LHcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation..”
Lodha and colleagues published their study in Biochemistry and Cell Biology – Biochimie Et Biologie Cellulaire (Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine beta-synthase. Biochemistry and Cell Biology – Biochimie Et Biologie Cellulaire, 2009;87(3):531-540).
For additional information contact:
S.M. Aitken
Carleton University
Dept. of Biology
Ottawa, ON K1S 5B6, Canada.
The publisher’s contact information for the journal Biochemistry and Cell Biology – Biochimie Et Biologie Cellulaire is: National Research Council Canada-N R C Research Press, Building M 55, Ottawa, ON K1A 0R6, Canada.