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

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Abstract
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:K m(MgATP)=1.0 mM,K i(ATP4−)=0.27 mM,K m(MnATP)=0.07 mM, andK i(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 affinityK 0.5(Ca2+)=0.02 mM,K 0.5(Mn2+)=3.8 mM,K 0.5(Mg2+)=4.7 mM, and an order of activity Mn2+>Mg2+>Ca2+. The data indicate that Mn2+ and Mg2+ ions may compete for a regulatory site distinct from the active site and increaseV m without changingK m(MgATP),K m(MnATP), orK i(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 cooperativityh 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+.

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