Stopped flow and steady state kinetic studies of the effects of metabolites on the soluble form of NADP+:isocitrate dehydrogenase.
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.
United States Department of Agriculture, Eastern Regional Research Center, Philadelphia, Pennsylvania 19118.
H M
HM
Farrell
J T
JT
Deeney
E K
EK
Hild
T F
TF
Kumosinski
D000818:Animals; D002417:Cattle; D002951:Citrates / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology; D008433:Mathematics; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding
D002951:Citrates; D007527:Isoenzymes; D009249:NADP; D007521:Isocitrate Dehydrogenase