The Origin of the Kinetic Isotope Effect and Mechanism of H/D Exchange in Radical C-C Bond Formation Catalyzed by Benzylsuccinate Synthase: Experiments and Microkinetic Modeling.

Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of (R)-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present the first microkinetic analysis of the full reaction, predicting kinetic isotope effects in the range of 2.587-2.604, close to the experimentally observed value (KIEexp= 2.13 ± 0.1). Our experiments confirmed that KIE values are lower in direct assays with substrate-saturated enzyme, relative to the values obtained for competitive kinetic isotope effects D(V/K) via substrate fractionation (3.69 ± 0.16). We postulate that this apparent KIE suppression originates from preferential binding of unlabeled versus labeled substrates to the enzyme, as well as from the rates of product release. We also show that tunneling effects have only minor influence on the observed KIE and D(V/K) values, although they have been estimated to potentially accelerate the overall reaction rate by approximately 17%. On the other hand, inclusion in the kinetic equation of the barrier recrossing effect, jointly with tunneling correction can result in a lowering of predicted KIE values to the range of 2.38-2.43. Furthermore, we analyze a slowly occurring, experimentally observed H/D exchange process in the product during incubation in D2O, confirming partial reversibility of the reaction. We estimate the rate of this H/D exchange and propose a potential mechanism. Our study contributes to elucidating the processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.