Phosphorylation of the calcium adenosine triphosphatase of sarcoplasmic reticulum: rate-limiting conformational change followed by rapid phosphoryl transfer

Abstract

The calcium adenosinetriphosphate of sarcoplasmic reticulum, preincubated with Ca2+ on the vesicle exterior (cE .cntdot. Ca2), reacts with 0.3-0.5 mM Mg .cntdot. ATP to form covalent phosphoenzyme (E .apprx. P .cntdot. Ca2) with an observed rate constant of 220 s-1 (pH 7.0, 25.degree. C, 100 mM KCl, 5 mM MgSO4, 23 .mu.M free external Ca2+, intact SR vesicles passively loaded with 20 mM Ca2+). If the phosphoryl-transfer step were rate-limiting, with kf = 220 s-1, the approach to equilibrium in the presence of ADP, to give 50% EP and kf = kr, would follow kobsd = kf + kr = 440 s-1. The reaction of cE .cntdot. Ca2 with 0.8-1.2 mM ADP proceeds to 50% completion with kobsd = 270 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from cE .cntdot. Ca2. The result is consistent with a rate-limiting conformational change of the cE .cntdot. Ca2 .cntdot. ATP intermediate followed by rapid (.gtoreq. 1000 s-1) phosphoryl transfer. Calcium dissociates from cE .cntdot. Ca2 .cntdot. ATP with kobsd = 80 s-1 and ATP dissociates with kobsd = 120 s-1 when cE .cntdot. Ca2 .cntdot. ATP is formed by the addition of ATP to cE .cntdot. Ca2. However, when E .cntdot. Ca2 .cntdot. ATP is formed in the reverse direction, from the reaction of E .apprx. P .cntdot. Ca2 and ADP, Ca2+ dissociates with kobsd = 45 s-1 and ATP dissociates with kobsd = 35 s-1. This shows that different E .cntdot. Ca2 .cntdot. ATP intermediates are generated in the forward and reverse directions, which are interconverted by a conformational change.