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

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.