A Kinetic Study of the Mechanism of Action of l-Gulonolactone Oxidase

From the ¹ From the Radioisotope Service, Wood Veterans Administration Center, and the Department of Biochemistry, Marquette University School of Medicine, Milwaukee, Wisconsin 53233
² From the Biochemical Section of the Oklahoma Medical Research Institute, Oklahoma City, Oklahoma 73104

The mechanism of action of l-gulonolactone oxidase has been studied by enzyme kinetic methods. Techniques have been developed for determining initial reaction rates above 1 atm of oxygen pressure.

The data are consistent with the following sequence of reactions: (a) combination of the enzyme with l-gulono--lactone; (b) reduction of the enzyme to produce ascorbic acid; (c) combination of the reduced enzyme with oxidant, which can be either oxygen or 2,6-dichloroindophenol (DCI); and (d) oxidation to the original state.

The substrate constants for l-gulono--lactone, oxygen, and DCI have been computed for the above mechanism by means of nonlinear regression methods.

In the presence of oxygen and DCI the enzyme decreased in activity as the oxygen pressure rose above 4 atm. This is consistent with there being two specific binding sites in close proximity to each other; each site binds only one of the two oxidants, oxygen or DCI, and inhibition occurs when both sites are occupied.

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