A pH switch affects the steady-state kinetic mechanism of pyranose 2-oxidase from Trametes ochracea

Abstract

The flavin-dependent pyranose 2-oxidase catalyzes the oxidation of d-glucose and other pyranoses at the C2 atom to yield 2-keto-sugars and hydrogen peroxide. Here, the steady-state kinetic mechanism of the enzyme from Trametes ochracea was investigated as a function of pH. Our findings show that the enzyme follows a bi-bi ping-pong kinetic mechanism at pH values <7.0, and a bi-bi ordered mechanism at pH values >7.0. Thus, at low pH the reactivity of the reduced enzyme with oxygen is controlled a by a conformational change of the enzyme that is associated with the release of the 2-keto-sugar from the active site of the enzyme. In contrast, at high pH the reduced enzyme–product complex permits the reaction of oxygen with the flavin. The study also illustrates that caution should be exerted in extrapolating the conclusions drawn on steady-state kinetic mechanisms established at a single pH value to other pH’s in flavoprotein oxidases.