The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes.
A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D010766:Phosphorylation
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium