Mechanisms for the oxidative decarboxylation of isocitrate: implications for control.

Abstract Mammalian tissues contain two isocitrate dehydrogenases: the DPN-dependent enzyme which is an oligomeric protein subject to allosteric activation by ADP, and the TPN-specific enzyme which is a monomeric protein not generally considered to be subject to regulation. Yet any controls exerted on the oxidative decarboxylation of isocitrate must necessarily encompass both enzymes since the reactions involve common substrates and metal ions. The two enzymes have been isolated in homogeneous state from porcine cardiac muscle. Similarity in their catalytic mechanisms is indicated by kinetic studies and evidence from chemical modification of enzymic functional groups. No appreciable deuterium isotope effect is observed when isocitrate-2- 2 H is substituted for isocitrate-2- 1 H as substrate for either the DPN- or TPN-dependent isocitrate dehydrogenase. The rates of both reactions are decreased about 5-fold in D 2 O as compared with H 2 O as solvent, implying that a proton transfer (perhaps from the hydroxyl group of isocitrate) may be involved in the rate-determining step. The pH dependence of V max measured at different temperatures suggests that an enzyme carboxylate ion is essential for the activity of both enzymes, although its pK is approximately 5.7 for the TPN enzyme and 6.6 for the DPN enzyme. This conclusion is strengthened by the inactivation of both enzymes by carbodiimide in the presence of nucleophiles. Similarly, chemical modification studies have implicated cysteinyl residues in the function of both enzymes. A single methionyl residue has been shown to be critical for catalytic activity of the TPN enzyme but a comparable residue has not yet been identified for the DPN enzyme. A lysyl residue has been shown to be directly involved only in the DPN enzyme. Mechanisms are proposed to account for catalysis of the oxidative decarboxylation of isocitrate by the two enzymes. Of the four stereoisomers, threo-D s -isocitrate is used as substrate by both isocitrate dehydrogenases. However, the pH dependence of K m for isocitrate suggests that dibasic isocitrate is the actual substrate for the DPN enzyme, whereas tribasic isocitrate is utilized by the TPN enzyme. Both enzymes require a divalent metal ion for activity. Kinetic and binding studies indicate that the TPN enzyme interacts with the preformed metal-tribasic isocitrate complex, whereas the DPN enzyme (depending on the available metal concentration) can either bind sequentially free metal ion followed by free dibasic isocitrate or can bind the preformed metal-dibasic isocitrate complex. Manganous ion produces the highest maximum velocity of each enzyme, but marked differences in the relative effectiveness of other metal ions, such as zinc and magnesium, are apparent. ADP, by binding to the allosteric site of the DPN enzyme, uniquely lowers the K m for dibasic isocitrate and its metal complex. However, other metabolic chelators which affect the distribution of free and metal-bound isocitrate can also indirectly alter the affinity of the substrate for both enzymes. The DPN- and TPN-specific isocitrate dehydrogenases are complementary. Several factors, including changes in the concentrations of different metal ions, subtle variations in pH or fluctuations in the levels of physiological chelators, influence the relative flux through the two pathways of oxidative decarboxylation of isocitrate. These changes may contribute to an alteration in the ratio of DPNH to TPNH in the intracellular environment.