Abstract
The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source 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. Stopped flow kinetics showed a distinct lag time, followed by attainment of an apparently linear final velocity. Direct nonlinear regression analyses of the reaction progress curves allowed for the calculation of the rate constant (kappa) for the transition of the enzyme from an inactive to an active form; this transition is best catalyzed by its metal-substrate complex. Preincubation with metal-substrate or metal-citrate nearly abolished the lag by increasing kappa 10-fold. 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 activate and inhibit the enzyme. This analysis suggested: (a) activation by binding to sites with an average dissociation constant of 0.25 mM; (b) inhibition by binding to sites with an average dissociation constant of 3.83 mM; and (c) modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+:isocitrate dehydrogenase.
Highlights
The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source 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
Direct nonlinear regression analyses of the reaction progress curves allowed for the calculation of the rate constant (k) for the transition of the enzyme from an inactive to an active form; this transition is best catalyzed by its metal-substrate complex
Examination of the early phases of the reaction progress curves in the stopped flow spectrophotometer showed a nonlinear response with a characteristic lag time prior to the achievement of a linear steady state
Summary
The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source 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. 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 activate and inhibit the enzyme This analysis suggested: (a) activation by binding to sites with an average dissociation constant of 0.25 mM; (b) inhibition by binding to sites with an average dissociation constant of 3.83 mM; and (c) modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. A survey of the distribution of Krebs cycle enzymes in mammary tissue showed that NADP+:isocitrate dehydrogenase (IDH)’ is predominately cytosolic (>90%) in nature, and that little or no NAD+:IDH activity is present [4] Since the latter enzyme is known to be allosterically regulated by metabolites and is thought to control Krebs cycle activity [5, 6], its absence in mammary gland prompted a study of the possible effects of metabolites on the kinetics of NADP’:IDH. Metal-substrate complex, metal ion, citrate, and metal-citrate complex on enzyme activity and activation were studied to gain further insight into possible mechanisms which may regulate this enzyme
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