Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase.

Toluene and related aromatic compounds represent an important class of pollutants that are long-lived in the environment and have relatively high water solubility. This is a particular problem in environments where oxygen is scarce, such as ground water supplies. It was initially thought that the only routes to degrade such compounds were through oxidative pathways that require molecular oxygen to functionalize otherwise inert aromatic hydrocarbons. However, the recent discovery in bacteria of anaerobic pathways for the degradation of aromatic compounds has aroused much interest, both for the potential they offer for bio-remediation in situations where oxygen is scarce, and because of the novel chemistry involved (1, 2). Various denitrifying and sulfate-reducing bacteria such as Thauera aromatica and Desulfobacula toluolica are able to live on toluene as their sole source of carbon and energy under anaerobic conditions (3, 4). The first step in this pathway is the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, as shown in scheme 1, in which hydrogen in the methyl group of toluene is retained in the product (5). This remarkable chemical transformation is catalyzed by benzylsuccinate synthase1 (BSS). Scheme 1 Reaction catalyzed by benzylsuccinate synthase BSS as a member of the growing class of glycyl radical-containing enzymes that includes pyruvate formate-lyase (PFL) and anaerobic ribonucleotide reductase (6-11). EPR studies show that the resting enzyme harbors an organic radical similar to that observed in these enzymes (12-14). Like other glycyl radical enzymes, BSS is extremely oxygen sensitive, and exposure to air results in oxidative cleavage of the α subunit at the site of the presumed glycyl radical (6). We recently investigated the stereochemistry of hydrogen transfer using deuterated toluene (15). We showed that when fumarate was the co-substrate, deuterium was transferred from toluene to the C-3 pro-(S) position of benzylsuccinate, implying that the addition of toluene to the double bond of fumarate was syn. However, when maleate (which is also recognized by the enzyme (16)) was the co-substrate, the addition of toluene occurs in an anti fashion. These observations provide evidence for the formation of the C-3 radical of benzylsuccinate as an intermediate, in which rotation about the C-2, C-3 bond can occur to relieve the sterically unfavorable cis conformation of the carboxylate groups when maleate is the co-substrate. Based on the observations above, the mechanism shown in Figure 1 is proposed. This involves the initial transfer of the glycyl radical to an active site cysteine residue, which then abstracts hydrogen from toluene to generate a benzylic radical. The benzylic radical subsequently undergoes addition to fumarate to generate the C-3 radical of (R)-benzylsuccinate. The abstracted hydrogen is transferred to the product to generate benzylsuccinate and the cysteinyl radical, and lastly the radical is transferred back to glycine. Figure 1 Proposed mechanism for benzylsuccinate synthase. The rapid loss of activity encountered during attempts to purify the enzyme and its extreme oxygen sensitivity (6) has severely hindered efforts to study the enzyme. As a result, virtually nothing is known experimentally regarding the kinetics or energetics of this highly unusual enzyme reaction. However, a density functional theoretical study has evaluated the BSS reaction and computed the relative stabilities of the radical intermediates and activation energies of each step in the putative mechanism (17). The rate-determining step in the reaction is predicted to be addition of the benzyl radical to fumarate, which is effectively irreversible (ΔG = -10.1 kcal/mol). To test this prediction and gain insight into the nature of the rate-determining step in BSS we have measured the deuterium kinetic isotope effects on Vmax and Vmax/Km for the addition of deuterated toluene to fumarate.