Open AccessChanges in the fluorescence of bound nucleotide during the reaction catalysed by glucose‐fructose oxidoreductase from Zymomonas mobilis
Author(s) -
HARDMAN Michael J.,
TSAO Marlene,
SCOPES Robert K.
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb16834.x
Subject(s) - chemistry , absorbance , zymomonas mobilis , reaction rate constant , fructose , fluorescence , oxidoreductase , photochemistry , conformational change , dissociation (chemistry) , dissociation constant , kinetics , enzyme , stereochemistry , chromatography , biochemistry , organic chemistry , ethanol , physics , receptor , ethanol fuel , quantum mechanics
The reduction of gluconolactone by glucose‐fructose oxidoreductase containing tightly bound NADPH (enzyme‐NADPH) is biphasic in nucleotide fluorescence. The initial rapid decrease, which represents quenching of the fluorescence by bound lactone, is followed by a slower decrease which corresponds to the change in absorbance. At low glucose concentrations, the oxidation of glucose by enzyme‐NADP + involves a single first‐order process with similar rate constants in fluorescence and absorbance. At higher glucose concentrations, the apparent first‐order rate constants for the fluorescence change are less than those for the absorbance change. This is consistent with a mechanism in which the fluorescence change occurs during the lactone dissociation step, which is slower than the hydrogen transfer step during which the absorbance change occurs. The rate constant for gluconolactone dissociation is 360 ± 10 s −1 and this step is therefore rate‐determining for the overall reaction. Reduction of fructose by enzyme‐NADPH is first order with a limiting rate constant of at least 2000 s −1 .