The glucose oxidase mechanism. Interpretation of the pH dependence.

Abstract The pH dependence of the steady state parameters of the glucose oxidase (EC 1.1.3.4, from Aspergillus niger) reaction was determined by O2-monitored experiments over the entire pH range from 3 to 10 at 25°, with d-glucose as substrate. The data were fitted to a three-parameter steady state rate equation and the significance of the steady state parameters was examined by stopped flow half-reaction and turnover measurements at the extremes of the pH range used. The major conclusions from these studies can be summarized as follows. 1. At low pH, in the presence of halide, the maximum turn-over number (kcat) is determined entirely by the rate of flavin reduction (k2) in the reductive half-reaction. Furthermore, substrate combines only with an unprotonated form of the oxidized enzyme and the reductive half-reaction can be represented as follows. H+ E0 (K1)/⇄/(H+) E0 + S (k1)/⇄/(k-1) E0 - S (k2)/→ Er + δ-lactone Since kcat and k2 are both specifically decreased by halides at low pH values, it is probable that the turnover rate in the low pH range is also limited by k2 in the absence of halide. The steady state absorption spectrum of E0 - S is indistinguishable from the spectrum of E0. This finding, together with the fact that removal of the 1-hydrogen from d-glucose is a ratelimiting process in flavin reduction is consistent with both a hydride transfer mechanism and with a flavin-glucose adduct mechanism in which this adduct is relatively unstable and never accumulates significantly as a kinetic intermediate. 2. The importance of k2 as a limiting first order process in turnover diminishes as the pH is raised. Thus, at pH 10 the major first order process in turnover is the breakdown of a species of oxidized enzyme, E'0, in the oxidative half-reaction. The rate of this process at pH 10.0 is 214 sec-1, whereas k2 has a value of 800 sec-1. 3. The reduced enzyme exists in two kinetically significant states of ionization, Er and Er-. The rapid reoxidation of Er with O2, to regenerate E0, is predominant at pH values less than 7. At pH values greater than 7, a much less rapid reaction of Er- with O2, leading to the formation of E'0-, becomes increasingly important. The species E'0- is unreactive with glucose and it is the conversion of a protonated form of E'0- to E0 which principally governs kcat at pH values greater than 7. We present a complete kinetic scheme describing the effects of pH and discuss the possible chemical significance of the species E'0.