title: Kinetic isotope effects as a probe of the beta-elimination reaction catalyzed by O-acetylserine sulfhydrylase.
abstract: Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies. Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate. The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively. Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0. D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH. The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments. Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Hwang
authors.forename: E U
authors.initials: EU
authors.lastname: Woehl
authors.forename: D E
authors.initials: DE
authors.lastname: Minter
authors.forename: M F
authors.initials: MF
authors.lastname: Dunn
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D002384:Catalysis; D003547:Cysteine Synthase / Q000378:metabolism*; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D015335:Molecular Probes; D012486:Salmonella typhimurium / Q000201:enzymology; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D014316:Tritium
chemical_list: D015335:Molecular Probes; D012545:Schiff Bases; D014316:Tritium; D012694:Serine; D003903:Deuterium; D003547:Cysteine Synthase