Catalytic Mechanism of Galactose Oxidase:  A Theoretical Study

Abstract

Density functional methods, alone and together with molecular mechanics, are used to study the catalytic mechanism of galactose oxidase. This enzyme catalyzes the conversion of primary alcohols to the corresponding aldehydes, coupled with reduction of dioxygen to hydrogen peroxide. It is shown that the proposed mechanism for this enzyme is energetically feasible. In particular the barrier for the postulated rate-limiting hydrogen atom transfer between the substrate and the tyrosyl radical, located at equatorial Tyr272, is very plausible. We propose that the radical site, prior to the initial proton transfer step, is located at the axial tyrosine (Tyr495). The radical is transferred to the equatorial tyrosine (Tyr272) simultaneously with the proton transfer. It is, furthermore, argued that the electron transfer from the ketyl radical intermediate to Cu(II) cannot be very exothermic, because this would render the oxygen reduction steps rate-limiting. Finally, the cysteine cross-link on the active site tyrosine is shown to have very minor effects on the energetics of the reaction.

How many ways to craft a cofactor?

“…In the case of the cysteine tyrosyl radical seen in galactose oxidase, the thioether at the ortho position of the tyrosyl radical does not appear to alter the redox potential appreciably (27) and may play primarily a structural role. In the case of the various quinones (Fig.…”

(N-Benzyl-bis-N′,N″-salicylidene)-cis-1,3,5-triaminocyclohexane copper(ii): a novel catalyst for the aerobic oxidation of benzyl alcohol

“…4, 5 In recent years there has been considerable focus on the preparation of small molecule models of the active site of GOase.…”

Homemade cofactors: Self-processing in galactose oxidase

“…This lowered potential nicely correlated with the enormous decrease in the oxidation potential of the Tyr-Cys crosslink from Ϸ1 V for regular tyrosines to 0.4 V in GAO (13). Whereas these reports suggested an electronic role for the crosslink, several other studies have presented support against this hypothesis with DFT calculations predicting only a 1.7 kcal͞mol stabilization of the radical because of the crosslink (14). These results have been interpreted to indicate a structural role of the thioether bridge.…”

“…Alternative mechanisms have also been proposed in which the Cys-Tyr⅐ abstracts a hydrogen atom from the alkoxide to generate a ketyl radical anion that then transfers an electron to Cu(II) (i.e., reversal of steps 2 and 3; ref. 14). Both steps may actually occur in an asynchronous concerted step depending on the substrate (32,33).…”