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The kinetics of pig heart triphosphopyridine nucleotide-isocitrate dehydrogenase. I. Initial velocity, substrate and product inhibition, and isotope exchange studies.

M. Uhr,V. Thompson,W. Cleland

Published 1974 in Journal of Biological Chemistry

ABSTRACT

Abstract Initial velocity, product and substrate inhibition, and isotope exchange studies are described which suggest that the kinetic mechanism of pig heart TPN-isocitrate dehydrogenase is random sequential with catalysis more rapid than product release. In the reverse reaction, substrate inhibition (presumably caused by bicarbonate), competitive versus TPNH and noncompetitive versus α-ketoglutarate, is seen at high CO2 levels, and more than one molecule of inhibitor can apparently combine with the enzyme. α-Ketoglutarate gives strong linear substrate inhibition which is uncompetitive versus TPNH or CO2, but the cause of this inhibition has not been established. Michaelis constants at pH 6.8, 25°, 2 mm Mg2+ are TPN, 0.25 µm; threo-ds-isocitrate, 1.1 µm; TPNH, 1.4 µm; α-ketoglutarate, 38 µm; CO2, 2.2 mm. TPN and TPNH are mutually competitive, whereas CO2 (or bicarbonate) and α-ketoglutarate are noncompetitive versus TPN and isocitrate. Isocitrate is competitive versus α-ketoglutarate and noncompetitive versus TPNH and CO2. TPN-TPNH exchange is not inhibited by high levels of TPN and TPNH, but shows substrate inhibition when isocitrate and α-ketoglutarate, CO2 and TPN, or isocitrate and TPNH are varied together. CO2-isocitrate exchange is also not inhibited by high TPN and TPNH, but substrate inhibition is seen when isocitrate and α-ketoglutarate are the varied reactants. In a random mechanism where catalysis is faster than reactant release, exchange between the varied reactants will be slower at low reactant levels than exchange between the other reactants with fixed levels, regardless which exchange is faster at high concentrations. The apparent Km values for the varied reactants will also be higher for exchange between them than for exchange between reactants at fixed high levels; these two criteria can be used to identify random mechanisms of this type. Preparations of pig heart TPN-isocitrate dehydrogenase from mitochondria, high speed supernatant of Potter-Elvehjem broken cells, and whole tissue acetone powder show only one activity band on gel electrophoresis at two pH values, and have identical kinetic properties, suggesting that only one isozyme exists in this tissue.

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