Abstract
The cytosolic form of NADP+ isocitrate dehydrogenase, a primary source of the NADP+ required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. The enzymatic reduction of NADP+ exhibits lag-burst (hysteretic) kinetics that are eliminated by the noncatalytic binding of the substrate, a complex (1:1) of a metal ion (Mn2+ or Mg2+) and isocitrate. Preincubation of the enzyme with metal-citrate complex also nearly abolished the lag or activation time. In steady-state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both stimulate and inhibit the enzyme. This analysis suggested hyperactivation by binding to sites with an average dissociation constant of .25mM, inhibition by binding to sites with an average dissociation constant of 3.83mM, and modulation (reactivation) by binding to sites with an average dissociation constant of 1.54mM. Conformational changes induced by the binding of ligands were assessed using circular dichroism. The results suggest that binding of metal-isocitrate induces a conformational transition involving tyrosyl residues that is related to the altered kinetic processes. Reexamination of Michaelis-Menten kinetics using nonlinear regression analysis also demonstrated hyperactivation of enzyme activity by metal-isocitrate with a dissociation constant equal to 21 μM (which is nearly seven times greater than the Michaelis constant). Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-iswitrate serve to modulate the activity of NADP+ isocitrate dehydrogenase.
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