An oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase has been isolated from Azotobacter vinelandii (ATCC 9104) cells. The enzyme is obtained in yields of 15-20% from unfractionated cell extracts. The purified enzyme appears to be homogeneous by the criteria of disc gel electrophoresis, ultracentrifugation, and gel filtration. It catalyzes the reduction of 120-135 μmoles of oxidized triphosphopyridine nucleotide/min per mg of protein under the assay conditions used. The purified enzyme is stable for several months in the frozen state. The enzyme has a molecular weight of 80,000, an S020,w of 4.6 X 10-13 sec, a D20,w of 5.4 X 10-7 cm2 sec-1, and an axial ratio of 5.0. By optical rotatory dispersion and circular dichroism measurements the native enzyme appears to have an α-helix content of about 30%. The enzyme does not dissociate into subunits in 6 M guanidine hydrochloride in the presence of reducing agents. The amino acid composition of the enzyme has been determined. It appears to have only three half-cysteine residues per mole of enzyme. The three thiol residues exhibit different degrees of reactivity. One of the thiol residues is readily titrated by 5,5′ dithiobis(2-nitrobenzoic acid), iodoacetic acid, and p-hvdroxymercuribenzoate. The titration of this reactive thiol group results in a complete loss of catalytic activity. The reactivity of a second thiol group is enhanced by titration of the first with p-hydroxymercuribenzoate. The third thiol residue appears to be buried within the protein molecule and is slowly titrated by p-hydroxymercuribenzoate. This thiol group becomes more reactive if the native enzyme is denatured with guanidine hydrochloride. The enzyme shows a high degree of specificity for oxidized triphosphopyridine nucleotide as electron acceptor; oxidized diphosphopyridine nucleotide has less than 1 % of the activity of oxidized triphosphopyridine nucleotide for accepting hydrogens from isocitrate. Oxidized deaminotriphosphopyridine nucleotide is incapable of serving as a substrate in the enzyme reaction. The purified enzyme is not markedly affected by a variety of adenine nucleoside derivatives which affect the oxidized diphosphopyridine nucleotide specific isocitrate dehydrogenases. The Michaelis constants for oxidized triphosphopyridine nucleotide and threo-uAsocitrate are 2.3 X 10-5 and 2.0 X 10-5 M, respectively. © 1969, American Chemical Society. All rights reserved.
