Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase

The reaction of benzylsuccinate
synthase, the radical-based addition
of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer
from a conserved cysteine to the nearby glycyl radical in the active
center of the enzyme. In this study, we analyze this step by comprehensive
computer modeling, predicting (i) the influence of bound substrates
or products, (ii) the energy profiles of forward- and backward hydrogen-transfer
reactions, (iii) their kinetic constants and potential mechanisms,
(iv) enantiospecificity differences, and (v) kinetic isotope effects.
Moreover, we support several of the computational predictions experimentally,
providing evidence for the predicted H/D-exchange reactions into the
product and at the glycyl radical site. Our data indicate that the
hydrogen transfer reactions between the active site glycyl and cysteine
are principally reversible, but their rates differ strongly depending
on their stereochemical orientation, transfer of protium or deuterium,
and the presence or absence of substrates or products in the active
site. This is particularly evident for the isotope exchange of the
remaining protium atom of the glycyl radical to deuterium, which appears
dependent on substrate or product binding, explaining why the exchange
is observed in some, but not all, glycyl-radical enzymes.