KINETICS AND THERMOSTABILITY OF NADP-ISOCITRATE DEHYDROGENASE FROM CEPHALOSPORIUM-ACREMONIUM

NADP-isocitrate dehydrogenase [isocitrate:NADP(+) oxidoreductase (deca rboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is ap parent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg2+ is the true substrate. Th e V-max depends on a basic form of an ionizable group of the enzyme-su bstrate complex with a pK(es) (pK of the enzyme substrate complex) of 6.9 and a Delta H-ion (activation enthalpy) of -2 +/- 0.4 kcal mol(-1) (ca. 8 +/- 2 kJ mol(-1)). The enzyme showed maximum activity at 60 de grees C, an unusually high temperature for a nonthermophilic fungus. T he thermodynamic parameters for isocitrate oxidative decarboxylation a nd for the binding of isocitrate and NADP(+) were calculated. We analy zed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40 degrees C following a first-order kinetics. T he presence of 12 mM Mg2+ pins 10 mM DL-isocitrate led to 100% protect ion of enzyme activity against inactivation at 60 degrees C for 120 mi n. Removal of either or both compounds led to activity loss. A greater stabilizing role for. Mg2+ was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.