Kinetics and Mechanism of Action of Glucose Oxidase

Abstract

The glucose oxidase from molds has been st.udied by a number of workers (l-8). The enzyme catalyzes the oxidation of n-glucose to n-glucono-b-lactone (7). Its substrate specificity has been examined, and it also attacks 2-deoxy-n-glucose, n-mannose, n-galactose, and n-xylose (5), although the turnover numbers for the last three sugars are very low. In this laboratory in recent years the formation of semiquinones in the oxidation and reduction of free flavins has been studied by spectrophotometry and electron spin resonance (9, lo), and attempts have been made to apply the results from model systems to flavin enzymes. No general relationship has been found between electron spin resonance signals and the appearance of absorption spectra corresponding to enzyme forms intermediate between fully oxidized and fully reduced flavin, nor do such oxidation-reduction intermediate forms necessarily play a part in catalysis (11). Each enzyme must therefore be examined separately, and the results of electron spin resonance and optical spectrophotometry correlated with rapid reaction studies, to establish whether or not intermediate forms of the enzyme react at rates sufficient to account for its catalytic activity. Electron spin resonance studies of glucose oxidase have already been reported by Beinert and Sands (12) and by Mason et al. (13), and while our own experiments on the glucose oxidase from AspergiZZus niger were in progress, Nakamura and Ogura (14) published an important paper on the kinetics of the oxidation of glucose by the enzyme from Penicillium arnagaskiense. Briefly, these papers indicate that semiquinoid intermediates play no part in the catalytic mechanism of glucose oxidase. This paper describes kinetic experiments by manometric and stopped flow methods with several substrates. Our experimental results confirm and extend those of Nakamura and Ogura (14), although our additional experiment.s suggest that a different interpretation is required to accommodate all the observations. Whereas Nakamura and Ogura identified a first order rate-limiting step with the conversion of enzyme-glucose complex to reduced enzyme and product, our results can be adequately explained without postulating the formation of an enzymeglucose complex, and we propose the dissociation of product from an enzyme-product complex as a first order rate-limiting step. With 2-deoxyglucose, however, there seems to be a typi-