Reversal of the extreme coenzyme selectivity of Clostridiumsymbiosum glutamate dehydrogenase

Reversal of the extreme coenzyme selectivity of Clostridiumsymbiosum glutamate dehydrogenase

Active-site mutants of glutamate dehydrogenase from Clostridiumsymbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) wi...

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Veröffentlicht in:The FEBS journal 2012-09, Vol.279 (17), p.3003
Hauptverfasser: Sharkey, Michael A, Gori, Alessandro, Capone, Marina, Engel, Paul C
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Bacteria
Biochemistry
Enzyme kinetics
Mutation
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Gori, Alessandro
Capone, Marina
Engel, Paul C
description Active-site mutants of glutamate dehydrogenase from Clostridiumsymbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) with retention of high levels of catalytic activity for the new coenzyme. For oxidized coenzymes, kcat/Km ratios of the wild-type and triple mutant enzyme indicate a shift in preference of approximately 1.6×107-fold, from 80000-fold in favour of NAD+ to 200-fold in favour of NADP+. For reduced coenzymes the corresponding figure is 1.7×104-fold, from 1000-fold in favour of NADH to 17-fold in favour of NADPH. A fourth mutation (N290G), previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant. [PUBLICATION ABSTRACT]
doi_str_mv 10.1111/j.1742-4658.2012.08681.x
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subjects Bacteria
Biochemistry
Enzyme kinetics
Mutation
title Reversal of the extreme coenzyme selectivity of Clostridiumsymbiosum glutamate dehydrogenase
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