title: Pre-steady-state kinetic analysis of enzyme-monitored turnover during cystathionine β-synthase-catalyzed H(2)S generation.
abstract: 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 (H(2)S)-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 H(2)S-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 H(2)S generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H(2)S 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 H(2)S.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI 48109-0600, USA.
authors.forename: Sangita
authors.initials: S
authors.lastname: Singh
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D003540:Cystathionine / Q000096:biosynthesis / Q000737:chemistry; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006710:Homocysteine / Q000737:chemistry / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D006710:Homocysteine; D003540:Cystathionine; D003541:Cystathionine beta-Synthase; D003545:Cysteine; D006862:Hydrogen Sulfide