期刊
FEBS JOURNAL
卷 279, 期 13, 页码 2296-2309出版社
WILEY
DOI: 10.1111/j.1742-4658.2012.08610.x
关键词
glucose-6-phosphate dehydrogenase; NAD(P) preference; NAD(P)H production; pentose phosphate pathway; progress curves analysis
资金
- Chilean Fondo Nacional de Desarrollo Cientifico y Tecnologico [FONDECYT 11080290]
- Chilean Ministry of Education [MECESUP 0713]
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 center dot 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 MichaelisMenten equation by including the effect of competitive product inhibition using the ?-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 (rRNAD) and NADP/NADPH (rRNADP). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rRNADP, but NADH production remains low within the range feasible for rRNAD. Nevertheless, we show that certain combinations of rRNADP and rRNAD sustain greater levels of NADH production over NADPH.
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