Kinetic behaviour of NADP-glutamate dehydrogenase from an extreme halophile Haloferax mediterranei in halophilic conditions (3 M KCl) and in glycerol

The kinetic mechanism of NADP-glutamate dehydrogenase EC 1.4.1.4) from the Archaeon Haloferax mediterranei was studied in 3 M KCI and in glycerol. Hal oferax mediterranei is a halophilic organism requiring 20-25% NaCl for opti mal growth, so its enzymes are stabilised by high salt concentrations. We h ave replaced the salt by 20% (v/v) glycerol in order to analyse if the kine tic properties and kinetic mechanism of the enzyme change at lower salt con centrations in order to apply the enzyme in a bioreactor. The kinetic mecha nism was studied by initial velocity measurements with, product and substra te analog inhibition. Product and substrates analog inhibition experiments indicate that (a), NADP(+) is a competitive inhibitor versus NADPH indicati ng that NADP(+) and NADPH both bind to free enzyme and (b), the binding ord er of substrates is NADPH, et-ketoglutarate and ammonium. The results sugge st that the mechanism in the amination reaction, with and without salt, is the same: sequential ordered with A (NADPH) binding in steady state and B ( alpha -ketoglutarate) in rapid equilibrium, with a dead-end (abortive) comp lex EC, and the last substrate bound is ammonium (C).