PHOSPHORYLATION OF THE CALCIUM-TRANSPORTING ADENOSINE-TRIPHOSPHATASE 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 0.25 mM LaCl3 and 0.3 mM [γ −32P]ATP with an observed rate constant of 6.5 s‒1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 °C). La•ATP binds to CE•Ca2 with a rate constant of 5 ✕ 106 M‒1s‒1, 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 0.34 and 65 s‒1, respectively, at saturating ADP (KSADP = 0.22 mM). 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 with 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, aE•Ca2•La•ATP, at 6.5 s‒1; this is much slower than the analogous conformational change at 220 s‒1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE•Ca2•La•ATP to cE•Ca2•La•ATP is 170 s−1. ATP does not dissociate measurably from aE•Ca2•La•ATP. Labeled EP formed from cE•Ca2 and La•ATP with leaky vesicles undergoes hydrolysis at 0.06 s‒1. 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. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE•Ca2 is mixed with 0.3 mM ATP and 1.0 mM CaCl2, the phosphoenzyme is formed with an observed rate constant of 3 s‒1. The phosphoenzyme formed from Ca•ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s‒1; there may be a small burst (α ≤ 0.05). © 1990 American Chemical Society. All rights reserved.