Kinetic isotope effects on the noncovalent flavin mutant protein of pyranose 2-oxidase reveal insights into the flavin reduction mechanism.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N(3) atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H(2)O(2) as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N(3)-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by d-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H(2)O(2). d-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases approximately 22-fold compared to that of the wild-type enzyme. The reduction of H167A using d-glucose isotopes (2-d-d-glucose, 3-d-d-glucose, and 1,2,3,4,5,6,6-d(7)-d-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of d-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the d-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the d-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters.