Kinetic Isotope Effects as a Probe of the β-Elimination Reaction Catalyzed by O-Acetylserine Sulfhydrylase

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Abstract
Primary and α-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 α-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-l-serine (OAS) to form the α-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 α-aminoacrylate intermediate compared to OAS being released from enzyme. The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of α-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 α-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 α-DKeq for the first half-reaction.