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.

Figure 1

Proposed mechanism for the reaction catalyzed by BSS

Figure 2

Representative HPLC chromatograms demonstrating the formation of HBS from BS and p-cresol. Inset: plot of reaction rate derived from integration of HBS peak areas.

Figure 3

Detection of d₈- and d₇-labelled toluene from the BSS-catalyzed exchange reaction of d₈-labeled BS with p-cresol. (A) GC trace; (B) mass spectrum of toluene peak.