title: Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase.
abstract: The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on Vmax and Vmax/Km when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on Vmax and Vmax/Km, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on Vmax and Vmax/Km that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688-7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: E Neil G
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D019755:Carbon-Carbon Lyases / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry*; D005650:Fumarates / Q000737:chemistry*; D007700:Kinetics; D014050:Toluene / Q000737:chemistry*
chemical_list: D005650:Fumarates; D014050:Toluene; D003903:Deuterium; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase