Pre-Steady-State Kinetic Analysis of Enzyme-Monitored Turnover during Cystathionine β-Synthase-Catalyzed H2S Generation

Cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals, i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H2S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457−22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H2S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H2S generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H2S and forms an aminoacrylate intermediate (k obs of 1.61 ± 0.04 mM−1 s−1 at low cysteine concentrations and 2.8 ± 0.1 mM−1 s−1 at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM−1 s−1 and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products, cystathionine and H2S.