The kinetics of glucose–fructose oxidoreductase from Zymomonas mobilis

Abstract

1. Glucose -fructose oxidoreductase operates by a classic ping-pong mechanism with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol. The K , values for these substrates were determined. The values of k,,, are 200 s-' and 0.8 s-' for the forward and reverse directions respectively.2. The overall catalytic process consists of two half-reactions with alternate reduction of NADP' and oxidation of NADPH tightly bound to the enzyme. Reduction of enzyme-NADP' by glucose and oxidation of enzyme-NADPH by gluconolactone involve single first-order processes. The values of the rate constants at saturating substrate are 2100 s-l and 8 s-' respectively; deuterium isotope effects indicate that these are for the hydrogen transfer step. Oxidation of enzyme-NADPH by fructose is first order with a limiting rate constant of at least 430 s-'. The reaction of enzyme-NADP' with sorbitol is biphasic, with rate constants for both phases less than 1 s-', This behaviour is explained by a mechanism in which the slow cyclisation of the acyclic form of fructose follows its dissociation from the enzyme.3. The rate-determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme-bound NADP' in the reverse direction.Following the discovery that the ethanol-producing bacterium Zymomonas mobilis produced sorbitol as well as ethanol when grown on sucrose or glucose/fructose mixtures [l -31, an enzyme was identified which catalysed the process [4, 51. The purified enzyme contains tightly bound NADP' and transfers hydrogens from glucose to fructose, producing gluconod-lactone and sorbitol [ 5 ] . The enzyme, named glucose -fructose oxidoreductase, is present in amounts up to 0.7% of the cytoplasmic protein; it was found to have low affinity for its substrates, but a high specific activity [ 5 ] . The presence of a gluconolactonase in Z . mobilis [5] ensures that the reaction is not reversible in vivo; thus the enzyme does not provide a route for metabolising sorbitol.Preliminary kinetic studies on the enzyme showed that the mechanism is ping pong, with a reduced NADP-enzyme intermediate. The K, for glucose was estimated to be between 6 -30 mM [4,5], highly dependent on fructose concentration, and the K , for fructose may be greater than 1 M.The ability to isolate the enzyme in large quantities, and a simple detection system for the reduced enzyme intermediate, have allowed us to carry out stopped-flow studies of the half-reactions and characterise the principal rate constants. A simple assay system for the reverse reaction has enabled us to obtain a detailed picture of the kinetic behaviour of the enzyme at pH 6.5. MATERIALS AND METHODSD-Glucose (predominantly a) was AR grade from Ajax Chemicals (Sydney, Australia). To ensure equilibration of the CI and B forms of D-glucose, stock glucose solutions were made up at least 18 h before use, unless otherwise stated below. Fructose (containing < 0.05% glucose), B-D-glucose (contain...

Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose‐fructose oxidoreductase from Zymomonas mobilis

“…The proposed mechanism for glucose-fructose oxidoreductase is shown in Scheme 1 [3]. The rate-determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme-NADP' in the reverse direction [3].Reactions catalysed by NAD(P)H-dependent enzymes can also be followed by monitoring the fluorescence of the nucleotide. For some dehydrogenases, for example lactate dehydrogenase [4], the fluorescence of enzyme-bound NADH depends on the nature of the intermediate and differs from that of free NADH.…”

“…The reduction of enzyme-NADP' by sorbitol was biphasic; the rate constants for the two processes were of similar magnitude and were less than 1 s-' . The proposed mechanism for glucose-fructose oxidoreductase is shown in Scheme 1 [3]. The rate-determining steps for the overall reaction are probably dissociation of gluconolactone in the forward direction and hydrogen transfer from sorbitol to enzyme-NADP' in the reverse direction [3].…”

“…The individual steps of the reaction of the enzyme with each of its substrates were studied, at 21 "C and pH 6.5, by monitoring the absorbance of enzyme-bound NADPH [3]. Reduction of enzyme-NADP' by glucose and oxidation of enzyme-NADPH by gluconolactone were first-order processes with rate constants at saturating substrate of 2100 s-' and 8 s-' respectively.…”

“…The reaction mechanism is ping pong [2, 31, with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol [3]. The overall reaction consists of two half-reactions with alternate reduction of the bound NADP' and oxidation of the NADPH [3].…”

“…The reaction mechanism is ping pong [2, 31, with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol [3]. The overall reaction consists of two half-reactions with alternate reduction of the bound NADP' and oxidation of the NADPH [3].The individual steps of the reaction of the enzyme with each of its substrates were studied, at 21 "C and pH 6.5, by monitoring the absorbance of enzyme-bound NADPH [3]. Reduction of enzyme-NADP' by glucose and oxidation of enzyme-NADPH by gluconolactone were first-order processes with rate constants at saturating substrate of 2100 s-' and 8 s-' respectively.…”