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The cofactor preference of glucose‐6‐phosphate dehydrogenase from Escherichia coli – modeling the physiological production of reduced cofactors
Author(s) -
Olavarría Karel,
Valdés Diego,
Cabrera Ricardo
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08610.x
Subject(s) - cofactor , nad+ kinase , dehydrogenase , glycerol 3 phosphate dehydrogenase , pentose phosphate pathway , enzyme , biochemistry , chemistry , redox , alcohol dehydrogenase , glucose 6 phosphate dehydrogenase , glycolysis , organic chemistry
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.