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 prope rties of the pyridoxal phosphate coenzyme that forms a series of intermedia tes in the reaction Of L-serine and L-homocysteine to form L-cystathionine. To characterize these reaction intermediates, we have carried out rapid-sc anning stopped-flow and single-wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluor escence spectroscopy under steady-state conditions. We find that the gem-di amine and external aldimine of aminoacrylate are the primary intermediates in the forward half-reaction with L-serine and that the external aldimine o f aminoacrylate or its complex with L-homocysteine is the primary intermedi ate in the reverse half-reaction with L-cystathionine. The second forward h alf-reaction of aminoacrylate with L-homocysteine is rapid. No primary kine tic isotope effect was obtained in the forward half-reaction with L-serine. The results provide evidence (1) that the formation of the external aldimi ne of L-Serine is faster than the formation of the aminoacrylate intermedia te, (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 ov erall reaction is rate-limited by the conversion of aminoacrylate to L-cyst athionine. We compare our results with cystathionine beta -synthase with th ose of related investigations of tryptopban synthase and O-acetylserine sul fhydrylase.