CaATP: the substrate, at low ATP concentrations, of Ca2+ ATPase from human erythrocyte membranes

Detailed kinetic analyses of ATP-splitting activities were obtained with purified Ca2+ ATPase from human erythrocyte membranes. At constant low total ATP levels increasing Mg2+ inhibited the activity of the enzyme, while increasing Ca2+ did not. Detailed analysis of this phenomenon with appropriate controls showed that this occurred because Ca2+ . ATP4- (CaATP) was the substrate for the enzyme: the inhibition by Mg2+ was due to the lowering of the CaATP concentration as Mg2+ replaced Ca2+ as the metal bound to ATP. Free ATP could not be the substrate, since its concentration could be lowered by increasing total Ca2+, and no inhibition of the reaction occurred. In addition to this site for CaATP, the enzyme may contain yet another metal-ATP site of much lower affinity and less specificity with respect to the metal. The data also indicate the presence of a Mg2+ site and a Ca2+ site in addition to the CaATP site. Since the enzyme also interacts with calmodulin, it effectively interacts with Ca2+ at three sites: the Ca2+ site, the CaATP site, and the Can-calmodulin site. The enzyme displays the same characteristics in the presence of calmodulin. However, the stimulation by calmodulin is always about three times higher in the presence of Mg2+ than in its absence. A model for the mechanism of this enzyme is discussed.