Catalytic mechanism of galactose oxidase: A theoretical study

Density functional methods, alone and together with molecular mechanics, ar e used to study the catalytic mechanism of galactose oxidase. This enzyme c atalyzes 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 partic ular the barrier for the postulated rate-limiting hydrogen atom, transfer b etween 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 radica l is transferred to the equatorial tyrosine (Tyr272) simultaneously with th e proton transfer. It is, furthermore, argued that the electron transfer fr om the ketyl radical intermediate to Cu(II) cannot be very exothermic, beca use this would render the oxygen reduction steps rate-limiting. Finally, th e cysteine cross-link on the active site tyrosine is shown to have very min or effects on the energetics of the reaction.