THERE IS ONLY ONE PHOSPHOENZYME INTERMEDIATE WITH BOUND CALCIUM ON THE REACTION PATHWAY OF THE SARCOPLASMIC-RETICULUM CALCIUM-ATPASE

Identical first-order rate constants for phosphorylation of the calciu m ATPase of sarcoplasmic reticulum by bound inorganic phosphate (P-i) of 25 +/- 2 s(-1) with empty vesicles and 25 +/- 1 s(-1) with vesicles that were passively loaded with 40 mM Ca2+ were obtained by treating the reaction as an approach to equilibrium (4 mM [P-32]P-i, 20 mM MgCl 2, 10 mM EGTA, and 100 mM KCl at pH 7.0 and 25 degrees C). The formati on of ADP-sensitive phosphoenzyme from P-i with Ca2+-loaded vesicles a lso proceeds with a first-order rate constant of 25 s(-1) and no detec table induction period. These identical rate constants show that lumen al Ca2+ does not inhibit the rate of phosphorylation of the enzyme by bound P-i and that there is no significant kinetic barrier for the con formational change that converts an ADP-insensitive to an ADP-sensitiv e phosphoenzyme intermediate with bound Ca2+. We conclude that there i s no evidence for the existence of two stable phosphoenzyme intermedia tes with bound Ca2+, such as E(1) similar to P.Mg.Ca-2 and Ca-2.E(2)-P .Mg, that are included in the E(1)-E(2) and related two-state models f or calcium transport by this enzyme. In general, coupling of a physica l reaction, such as muscle contraction or vectorial transport, to a ch emical reaction, such as ATP hydrolysis, requires more than two states in the reaction cycle. It is not yet clear how the driving force that is provided by the movement of two Ca2+ ions from low-affinity to hig h-affinity sites is utilized to bring about ATP synthesis at a rate th at is too fast to measure, when ADP is added to phosphoenzyme in Ca2+- loaded vesicles.