The kinetics of glucose-fructose oxidoreductase from Zymomonas mobilis.
Glucose-fructose oxidoreductase operates by a classic ping-pong mechanism with a single site for all substrates: glucose, fructose, gluconolactone and sorbitol. The Km values for these substrates were determined. The values of kcat are 200 s-1 and 0.8 s-1 for the forward and reverse directions respectively. 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-1 and 8 s-1 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-1. The reaction of enzyme-NADP+ with sorbitol is biphasic, with rate constants for both phases less than 1 s-1. This behaviour is explained by a mechanism in which the slow cyclisation of the acyclic form of fructose follows its dissociation from the enzyme. 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.
Hardman
M J
MJ
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Scopes
R K
RK
D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones; D010084:Oxidation-Reduction; D013012:Sorbitol / Q000378:metabolism; D013379:Substrate Specificity
D005942:Gluconates; D007783:Lactones; D005632:Fructose; D013012:Sorbitol; D002237:Carbohydrate Dehydrogenases; C050020:glucose-1-fructose-2-oxidoreductase; D005947:Glucose; C010730:beta-glucono-1,5-lactone