Properties of an NADP+-specific isocitric dehydrogenase from Thiobacillus novellus

Abstract

Isocitric dehydrogenase from Thiobacillus novellus has been purified 12-fold and some of its properties were studied. The enzyme is NADP+-specific and shows an absolute requirement for a divalent cation. Mn2+ was the most effective of those tested but was replaceable to varying degrees by any of several other divalent cations. The order of effectiveness at a concentration of 3.3 × 10−5 M was Mn2+ > Co2+ > Zn2+ > Mg2+ > Ni2+ > Hg2+ > Pb2+ > Ca2+. The optimal pH was about 8.0 with both potassium phosphate and Tris–HCl buffers. Arsenate and phosphate were capable of increasing enzyme activity in the presence of low concentrations of buffer. Added phosphate resulted in maximal activity at a concentration of 40 mM. Double reciprocal plots of velocity against Pi concentration gave a [Formula: see text] of 0.38 mM. The reaction was not reversible under any of the conditions of the investigation, nor was activity enhanced by AMP. In fact, depending upon the concentration used, most of the nucleotides tested were inhibitory. The order of effectiveness was mononucleotide < dinucleotide < trinucleotide. Of interest is the observation that the enzyme was inhibited by low concentrations of oxalacetate and a concerted effect was demonstrated by glyoxylate and oxalacetate.