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.

SUBMITTER: Li L 

PROVIDER: S-EPMC2527060 | biostudies-other | 2006 Dec

REPOSITORIES: biostudies-other