The pH dependence of the individual steps in the glucose oxidase reaction.

Abstract The kinetics of oxidation of d-mannose, 2-deoxy-d-glucose, and d-glucose by glucose oxidase from Penicillium notatum has been studied in the pH range from 3 to 8 at 25°, in the presence of 0.2 m KCl. The pH dependence of the individual steps in the catalytic mechanism was determined by stopped flow spectrophotometric measurements of each half-reaction, in conjunction with conventional steady state kinetic measurements of the over-all reaction. The following scheme, originally deduced by Gibson, Swoboda, and Massey (J. Biol. Chem., 239, 3927 (1964)) for the enzyme from Aspergillus niger at pH 5.6 and 25°, was found to accommodate the results at any value of pH in the range examined. [see PDF for equation] where Eo, Er, S, and P are, respectively, oxidized enzyme, reduced enzyme, substrate, and product. The pH dependence and other characteristics of each step in turn are as follows. 1. The pH profile for k1 is sigmoid, and indicates that the combination of substrate with Eo is dependent upon a basic group (pK1 = 5.00 for mannose and glucose; pK1 = 5.35 for 2-deoxyglucose) in the enzyme. There is a small solvent deuterium isotope effect on k1. Halides have a specific effect on pK1, causing it to increase markedly, but have no effect on k1. 2. Flavin reduction, controlled by k2 and measured with 2-deoxyglucose, is relatively insensitive to pH in the range from 3 to 8. Halides also specifically decrease the rate of flavin reduction. 3. The pH profile for k4 is sigmoid and indicates that the combination of O2 with Er is dependent upon an acidic group (pK4 = 6.90) in the enzyme. 4. The pH profile for k5 is bell shaped and indicates that this terminal, first order process (which may consist of more than one step) is dependent upon an acidic (pK'5 = 7.40) and a basic (pK5 = 4.10) group in the enzyme. Based on these findings, a kinetic scheme is presented which accounts for the pH dependence of the steady state velocity of oxidation of the three sugars in the pH range from 3 to 8. Analogue simulation of the partition of the enzyme between oxidized and reduced forms during turnover at different values of pH, with experimentally determined values of acid dissociation and rate constants, agreed with turnover patterns obtained in stopped flow spectrophotometric experiments at 450 mµ.