The Reaction of Yeast Cystathionine β-Synthase Is Rate-Limited by the Conversion of Aminoacrylate to Cystathionine

Our studies of the reaction mechanism of cystathionine β-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 α-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 β-synthase with those of related investigations of tryptophan synthase and O-acetylserine sulfhydrylase.