Mechanistic studies on glucose-fructose oxidoreductase from Zymomonas mobilis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in biochemistry at Massey University, New Zealand

Abstract

The reaction mechanism catalysed by glucose-fructose oxidoreductase was studied in detail by comparing the presteady kinetic results from absorbance and fluorescence experiments using the stopped-flow apparatus. The oxidation of enzyme-NADPH by gluconolactone was a biphasic reaction, with a fluorescence decrease within the dead time, followed by a slower decrease which corresponds to the absorbance change. This behaviour was due to quenching of the fluorescence of enzyme-NADPH by bound gluconolactone. In both absorbance and fluorescence experiments, reduction of enzyme­ NADP+ by glucose appeared to be a single first order process. At high glucose concentrations the rate constants for absorbance were higher than those for fluorescence, with limiting values of 2100 ± 130 s-1 and 373 ± 14 s-1, respectively, under similar conditions. The fluorescence change for the glucose/gluconolactone half reaction occurs during dissociation of gluconolactone, while the absorbance change represents hydrogen transfer from glucose to enzyme-NADP+.

The oxidation of enzyme-NADPH by fructose was shown to involve one phase in both absorbance and fluorescence. The reduction of enzyme-NADP+ by sorbitol was shown to be biphasic in both absorbance and fluorescence. However, the two phases in the sorbitol reaction are not well separated, because the difference between the two rate constants is small, and the rate constants were difficult to determine.

Glucose-fructose oxidoreductase was unusually stable to temperature changes and unaffected by reactive dye binding, but unstable during dialysis. The NADP+ was shown to be essential for the integrity of the enzyme, since GFOR was denatured upon removal of NADP+ from this enzyme and this denaturation was irreversible. Preliminary inhibitor studies with diethyl pyrocarbonate suggested that histidine residues may be important in the catalytic reaction of GFOR.