A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4-, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP4- (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained: Km(MgATP) = 1.0 mM, Ki(ATP4-) = 0.27 mM, Km(MnATP) = 0.07 mM, and Ki(CaATP) = 0.015 mM. MgATP, MnATP, ATP4-, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP4- from MgATP. Ca2+ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinity K0.5(Ca2+) = 0.02 mM, K0.5(Mn2+) = 3.8 mM, K0.5(Mg2+) - 4.7 mM, and an order of activity Mn2+ greater than Mg2+ greater than Ca2+. The data indicate that Mn2+ and Mg2+ ions may compete for a regulatory site distinct from the active site and increase Vm without changing Km(MgATP), Km(MnATP), or Ki(ATP4-). The interactions of ATP4- and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg2+ and Mn2+, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg2+ nor Mn2+, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP4- and CaATP are involved in dead-end inhibition. For MgCl2 saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativity h from a basal value of 2.6, when Mg2+ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg2+.