The reaction of yeast cystathionine beta-synthase is rate-limited by the conversion of aminoacrylate to cystathionine.
Our studies of the reaction mechanism of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme that forms a series of intermediates in the reaction of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-scanning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. We find that the gem-diamine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine of aminoacrylate or its complex with L-homocysteine is the primary intermediate in the reverse half-reaction with L-cystathionine. The second forward half-reaction of aminoacrylate with L-homocysteine is rapid. No primary kinetic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimine of L-serine is faster than the formation of the aminoacrylate intermediate, (2) that aminoacrylate is formed by the concerted removal of the alpha-proton and the hydroxyl group of L-serine, and (3) that the rate of the overall reaction is rate-limited by the conversion of aminoacrylate to L-cystathionine. We compare our results with cystathionine beta-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.
Jhee
K H
KH
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
Niks
D
D
McPhie
P
P
Dunn
M F
MF
Miles
E W
EW
D003540:Cystathionine / Q000096:biosynthesis*; D003541:Cystathionine beta-Synthase / Q000378:metabolism*; D005246:Feedback; D006710:Homocysteine / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013053:Spectrophotometry
D006710:Homocysteine; D003540:Cystathionine; D012694:Serine; D003541:Cystathionine beta-Synthase