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.

Scheme 1

Reaction catalyzed by benzylsuccinate synthase

Figure 1

Proposed mechanism for benzylsuccinate synthase.

Figure 2

HPLC assay of BSS activity in cell-free extracts of T. aromatica. Top Typical HPLC chromatograph with position of the benzylsuccinic acid peak indicated. Bottom Enlargement of the region between 10 and 13 min illustrating the time-dependent increase in benzylsuccinic acid.

Figure 3

Determination of the isotope effect on V_max for BSS. Initial velocity plots for BSS, reacting with saturating concentrations of toluene. The symbols represent the reactions of unlabelled toluene (triangles) or d₈-toluene (squares) at 4 °C (closed symbols) or 30 °C (open symbols).

Figure 4

Determination of V/K isotope effect for BSS Top Representative electrospray mass spectra (negative mode) showing the ratio of benzylsuccinate (M-1 = 207) to d₈-benzylsuccinate (M-1 = 215) produced by incubation of BSS with a 1:1 mixture of toluene and d₈-toluene at 30 °C. Bottom Determination of the isotope effect on V_max/K_m by extrapolation of the apparent isotope effect to zero extent of reaction. Data shown for 4 °C (□)or at 30 °C (○).

Figure 5

Free energy profile for the BSS reaction calculated by density functional theory. The data used to construct the figure are taken from reference (17). The heights of the energy barriers (kcal/mol) are indicated by the side of each barrier; the bold numbers refer to the enzyme species shown in Figure 1; the steps subject to a deuterium isotope effect are indicated by dashed lines.

Figure 6

Qualitative free energy profile for the initial steps in the BSS-catalyzed reaction that is consistent with the experimentally observed isotope effects on V_max and V_max/K_m. The numbers refer to the enzyme species shown in Figure 1; the step subject to a deuterium isotope effect is indicated by dashed lines.