title: The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli--modeling the physiological production of reduced cofactors.
abstract: In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Karel
authors.initials: K
authors.lastname: Olavarría
authors.forename: Diego
authors.initials: D
authors.lastname: Valdés
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cabrera
mesh_terms: D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology*; D019298:Glucose-6-Phosphate / Q000737:chemistry* / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase