title: Mechanism of benzylsuccinate synthase probed by substrate and isotope exchange.
abstract: Benzylsuccinate synthase catalyzes the first step in the anaerobic metabolism of toluene through an unusual reaction in which toluene is added to fumarate to produce (R)-benzylsuccinate. We have exploited the broad substrate range of the enzyme to demonstrate that the enzyme can catalyze the exchange of p-cresol with the benzyl portion of benzylsuccinate to form (4-hydroxybenzyl)-succinate, indicating that the reverse reaction (disproportionation of benzylsuccinate to toluene and fumarate) must have occurred. Even though the equilibrium constant for the reverse reaction is extremely unfavorable, Keq approximately 8 x 10-11 M at 4 degrees C, the enzyme catalyzes the reverse reaction at a rate only 250-fold slower than the forward reaction. Furthermore, using deuterium-labeled benzylsuccinate we observe partial exchange of deuterium with the solvent. This provides the first direct evidence that the migrating hydrogen is transferred to a labile site on the protein during catalysis, which is consistent with the participation of the proposed active-site cysteine residue in the mechanism of BSS.
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 / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D007554:Isotopes / Q000737:chemistry; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D005609:Free Radicals; D007554:Isotopes; D019755:Carbon-Carbon Lyases; C114354:benzylsuccinate synthase