The kinetics of NAD-linked isocitrate dehydrogenase from calf heart

Abstract

From kinetic data, free manganous ion, free dibasic isocitrate and the manganese-dibasic isocitrate complex have been identified as the forms of substrate which bind to NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from calf heart. A kinetic model is proposed in which the active enzyme-substrate complex may be formed by any of three binding pathways, the predominant pathway depending upon the relative magnitude of the concentrations of substrates and the dissociation constants. Dissociation constants for the active enzyme-substrate complex and its intermediate forms have been determined at pH 6.1 and 7.1. ADP has been observed to lower significantly the Michaelis constants for manganous and isocitrate species while having no effect on maximal velocity. The p K es of an essential ionizable amino acid residue has been found to be 6.51, 6.47 and 6.43 at 12, 22 and 32 °C, respectively. The heat of ionization of this residue has been determined to be ΔH i = +1.56 kcal/mole. On the basis of the above data and analogies to other enzymes, this essential amino acid residue is suggested to be either glutamyl or aspartyl. The activation energy of the enzyme catalyzed reaction was found to be E a = 12.6 kcal/mole.