Abstract
Abstract In order to trace the pathway of substrate-derived hydrogen in the mechanism of the glucose oxidase reaction, the kinetics of oxidation of d-glucose-1-2H was studied by conventional and stopped flow spectrophotometric methods. The results were compared with those for d-glucose-1-1H. A deuterium kinetic isotope effect was shown in the over-all reaction kinetics. The magnitude of this effect was highly dependent on the concentrations of sugar and oxygen. Stopped flow kinetic studies of the glucose half-reaction under anaerobic conditions located the kinetic isotope effect (between 10 and 15 at 3°) at the flavin reduction step. These studies also provided evidence for a complex between oxidized enzyme and d-glucose. Stopped flow measurements of the enzyme in turnover with d-glucose-1-2H showed that both the turnover rate of the enzyme at infinite oxygen concentration and the turnover rate of the oxidized fraction of the enzyme were almost identical with the independently measured rate of the d-glucose-1-2H half-reaction. These results indicated that, on the substitution of deuterium for hydrogen, no first order rate-limiting steps could be shown, under the conditions of the experiments, other than that associated with flavin reduction by d-glucose-1-2H. Our kinetic results are accomodated by the following general scheme. [see PDF for equation] where P1 and P2 are d-glucono-δ-lactone and H2O2, respectively, and G is β-d-glucose. The change in the rate constant for flavin reduction brought about by substrate deuteration requires the results to be expressed by two special cases of the general steady state rate equation. For d-glucose-1-1H, as was shown previously, [see PDF for equation] whereas for d-glucose-1-2H [see PDF for equation] These rate equations express both the substrate concentration dependence of the kinetic isotope effect on the over-all reaction kinetics and the fact that the isotope effect on the flavin reduction step is much larger than that observed in the overall reaction. All rate constants in the scheme were evaluated, although there was an uncertainty of about ±25% in the value of k2 for d-glucose-1-1H. The validity of both the scheme and the numerical values of the rate constants was checked satisfactrorily by analogue computer simulation of the results of the stopped flow experiments performed on the glucose half-reaction and the enzyme in turnover. Because of the dominance of k2 in the kinetics of oxidation of d-glucose-1-2H, it was not possible to determine whether hydrogen transfer was important in the oxygen half-reaction. There was no solvent deuterium effect on any step in the mechanism with either substrate.
Highlights
In order to trace the pathway of substrate-derived hydrogen in the mechanism of the glucose oxidase reaction, the kinetics of oxidation of D-glucose-l-2H was studied by conventional and stopped flow spectrophotometric methods
When the glucose-l‘H data are interpreted in terms of Equation 1, and noting the fact that/e, >> k5 at this temperature (a), the agreement between the present results and those of Gibson et al, obtained from stopped flow experiments, is good. It is apparent from a comparison of the glucose-l-IH and glucose-l-2H rate data in Fig. 1 that a considerable deuterium kinetic isotope effect is observed in the over-all reaction kinetics and that the magnitude of the observed effect is highly dependent on the concentrations of glucose and oxygen
With glucose-lJH, the enzyme is predominantly in the oxidized form during the steady state period of the reaction and the time required for exhaustion of the oxygen is much longer than it is with glucose-1JH. These results indicate that the slowing of the reductive half-reaction by a deuterium kinetic isotope effect is sufficient to make the reductive half-reaction a dominant factor in the reaction kinetics, except at very low oxygen concentrations
Summary
In order to trace the pathway of substrate-derived hydrogen in the mechanism of the glucose oxidase reaction, the kinetics of oxidation of D-glucose-l-2H was studied by conventional and stopped flow spectrophotometric methods. Stopped flow kinetic studies of the glucose half-reaction under anaerobic conditions located the kinetic isotope effect (between 10 and 15 at 3O) at the flavin reduction step. These studies provided evidence for a complex between oxidized enzyme and D-glucose. D-glucose-l-2H showed that’ both the turnover rate of the enzyme at infinite oxygen concentration and the turnover rate of the oxidized fraction of the enzyme were almost identical with the independently measured rate of the D-ghcose-l-2H half-reaction These results indicated that, on the substitution of deuterium for hydrogen, no first order rate-limiting steps could be shown, under the conditions of the experiments, other than that associated with flavin reduction by D-ghCOSt?-l-2H. An important consequence of this change in rate-determining step is the fact that complex formation between oxidized enzyme and n-glucose-l-2H can be clearly shown
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