Abstract
Glucose-6-phosphate dehydrogenase (G6PD) oxidizes glucose-6- phosphate (G6P) to 6-phosphoglucono-d-lactone (6PGL) and reduces nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH. Detailed studies of human G6PD are hindered by difficulties in purifying significant quantities of this enzyme for initial-rate procedures. In addition, the instability of 6PGL prevents a kinetic analysis of the reverse reaction. A novel method of investigating enzymatic properties by visual comparison of mechanism plots with experimental progress curves is proposed here. Selwyn's test was used to rule out the possibility of enzyme inactivation, thus making possible the use of progress curve analysis. A computer spreadsheet was devised to graph three possible ternary mechanisms, random bi-bi, Theorell-Chance (T-C) and ordered bi-bi, on the same system of coordinates with experimental reaction data. Each graph was plotted by Euler's method of numerical integration from corresponding ordinary differential equations describing the cited mechanisms, while the progress curves were obtained by monitoring the absorbance of NADPH at 340 nm. The Solver regression module was used to simultaneously fit 11 simulated curves (repeated for each mechanism) against 11 different experimental progress curves obtained by UV/VIS spectroscopy. This process generated three complete sets of kinetic parameters, which were further analyzed statistically using a Monte-Carlo simulation. The magnitude of the standard errors and simplicity considerations indicate that a T-C mechanism best explains the experimental data.
