Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: Rapid phosphoryl transfer following a rate-limiting conformational change

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE·Ca2) is phosphorylated upon the addition of LaCl3 and [γ-32P]ATP with an observed rate constant of 6.5 s-1. La·ATP binds to cE·Ca2 while ATP, Ca2+, and La3+ dissociate from cE·Ca2·La·ATP at ≤1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La·ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP·ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (α) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.35 and 65 s-1, respectively, at saturating ADP. The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE·Ca2 by La·ATP at 6.5 s-1 and the kinetics for the reaction of EP and ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE·Ca2·La·ATP to the form of the enzyme that is activated for phosphoryl transfer. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg·ATP and La·ATP; however, the strong binding of La·ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La·ATP