Activation of NADP-specific isocitrate dehydrogenase by chelating agents

Abstract

The effects of different metal chelating agents on the activity of the NADP-linked isocitrate dehydrogenase from pig heart have been studied. Addition of ethylene glycolbis(β-aminoethyleter) N,N′-tetraacetic acid, N-hydroxyethylenediamine triacetic acid, and ethylenediamine tetraacetic acid (EDTA) under certain conditions could enhance the activity by a factor of nearly 3. Moreover, the time lag occurring before the reaction rate approached a constant value at suboptimal metal-ion concentrations was abolished by the metal chelating agents. S 0.5 for isocitrate increased slightly in the presence of the metal-chelating agents. The substrate inhibition occurring at high NADP concentrations was abolished by the activator. The pH optimum was the same in the absence and presence of EDTA. The extent of activation increased on a relative basis with increasing pH. Studies of the sedimentation behavior of the enzyme under different conditions suggested that the effect of the metal-chelating agents could not be accounted for by aggregation or depolymerization of the enzyme. NADPH affects the enzyme activity in a similar way, although less efficiently than the metal chelating agents. The results indicate that most organic metal complexes can activate the enzyme. It has previously been suggested that isocitrate complexed with a metal ion is the real substrate for the enzyme. If this holds true, the activation found with other organic metal complexes can be accounted for by a reduction in the apparent K m for the isocitrate metal complex and by an increase in the maximum rate of the reaction by removal of the substrate inhibition at high NADP concentrations.