Effect of a conservative mutation of an active site residue involved in substrate binding on the hydride tunneling reaction catalyzed by choline oxidase

The reaction of alcohol oxidation catalyzed by choline oxidase was previously shown to occur through the tunneling of a hydride ion from the α-carbon of an activated alkoxide species to the N(5) atom of FAD within a highly preorganized enzyme-substrate complex [G. Gadda, Biochemistry 47 (2008) 13745-13753]. In the present study, a glutamate residue (312) that participates in substrate binding has been replaced with aspartate by site-directed mutagenesis, and the effect of the substitution on the kinetic parameters of the enzyme and the mechanism of hydride ion transfer were investigated using stopped-flow spectrophotometry. The thermodynamic parameters associated with the enzymatic reaction catalyzed by the mutant enzyme and the temperature dependence of the kinetic isotope effects are consistent with a hydride transfer reaction occurring either through environmentally assisted tunneling or a classical over-the-barrier transition state, but not within a preorganized enzyme-substrate complex. In contrast, less than threefold changes in the Kd values for choline were observed in the mutant enzyme with respect to the wild-type enzyme. Thus, the conservative substitution of an active site residue that is involved in substrate binding, but not directly in catalysis, affects primarily the kred value that reports on catalysis and minimally the Kd value that reports on binding, thereby providing an example of the interplay that these kinetic parameters may have in enzymatic reactions.