REGULATION OF THE SOLUBLE FORM OF NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE-SPECIFIC ISOCITRATE DEHYDROGENASE FROM LACTATING BOVINE MAMMARY-GLAND - EFFECTS OF METABOLITES ON ACTIVITY AND STRUCTURE

The cystolic form of NADP(+): isocitrate dehydrogenase, a primary sour ce of the NADPH 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(+) exhibit s lag-burst (hysteretic) kinetics that are eliminated by the noncataly tic binding of the substrate, a complex (1:1) of a metal ion (Mn2(+) o r Mg2(+)) and isocitrate. Preincubation of the enzyme with metal-citra te complex also nearly abolished the lag or activation time. In steady -state experiments, experiments, analyses of velocity versus metal-cit rate complex as a binding isotherm, following the assumptions of Wyman 's theory of thermodynamic linkage, showed that binding of metal-citra te complex could both stimulate and inhibit the enzyme. This analysis suggested hyperactivation by binding to sites with an average dissocia tion constant of 3.83 mM, and modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Conformational changes induced by the binding of ligands were assessed using circula r dichroism. The results suggest that binding of metal-isocitrate indu ces a conformational transition involving tyrosyl residues that is rel ated to the altered kinetic processes. Reexamination of Michaelis-Ment en kinetics using nonlinear regression analysis also demonstrated hype ractivation of enzyme activity by metal-isocitrate with a dissociation constant equal to 21 mu M (which is nearly seven times greater than t he Michaelis constant). Concentration ranges observed for these transi tions are compatible with physiological conditions, suggesting that co mplexes of metal-citrate and metal-isocitrate serve to modulate the ac tivity of NADP(+): isocitrte dehydrogenase.