Residue N84 of yeast cystathionine beta-synthase is a determinant of reaction specificity.
Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The kcat/KmL-Ser of the N84A, N84D and N84H mutants for the beta-replacement reaction is reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal beta-elimination activity was detected for N84D (kcat/KmL-Ser=0.23+/-0.02 M(-1) s(-1)) and N84H (kcat/KmL-Ser=0.34+/-0.06 M(-1) s(-1)), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with L-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site.
Lodha
Pratik H
PH
Department of Biology, Carleton University, Ottawa, Canada K1S 5B6.
Hopwood
Emily M S
EM
Manders
Adrienne L
AL
Aitken
Susan M
SM
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D003540:Cystathionine / Q000737:chemistry; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D006710:Homocysteine / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D006710:Homocysteine; D001224:Aspartic Acid; D003540:Cystathionine; D006639:Histidine; D014364:Tryptophan; D003541:Cystathionine beta-Synthase; D000409:Alanine