Allosteric Inhibition of a Triphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase from Crithidia fasciculata by Nucleoside Triphosphates

Abstract

Abstract A soluble TPN+-specific isocitrate dehydrogenase has been isolated from Crithidia fasciculata. Although this enzyme obeys classical Michaelis-Menten kinetics, it has been shown to be inhibited by nucleoside triphosphates. A kinetic study, with ATP as a model inhibitor, indicated that the nucleotide acted at a locus separate from the catalytic site of the enzyme. Both the purine or pyrimidine and a β and γ-phosphate group were required for inhibition of the enzyme, since nucleoside mono- and diphosphates were not inhibitory. The nucleotides did not compete with either isocitrate or TPN+ for the active site; the effect of the nucleotide was upon the Vmax for both substrate and coenzyme rather than on their apparent Km. Two molecules of ATP appeared to be required for inhibition. The inhibition was found to be of a cumulative type when all four trinucleotides were used. Studies on the changes in entropy and free energy of the reaction, although not conclusive, indicated that the protein was probably capable of undergoing a physical change during combination with the nucleotide inhibitor. It is suggested that allosteric modification of enzymes which do not exhibit sigmoid rate-substrate relationships may be a more general phenomenon than is realized.