Reactions of the sarcoplasmic reticulum calcium adenosine triphosphatase with adenosine 5'-triphosphate and calcium that are not satisfactorily described by an E1-E2 model

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Abstract
Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 .+-. 7 s-1 after free enzyme was mixed with saturating ATP and 50 .mu.M Ca2+; this is one-third the rate constant of 220 s-1 for phoshorylation of enzyme preincubated with calcium, cE .cntdot. Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25.degree. C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives .apprx. 50% E .apprx. P .cntdot. Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE .cntdot. Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of > 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid (EGTA) show that the rate constants for dissociation of [.gamma.-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E .cntdot. ATP .cntdot. Ca2+ and at 24 s-1 from E .cntdot. ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 .mu.M ATP and 50 .mu.M Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (Kb). The fraction of Ca2+-free E .cntdot. [.gamma.-32P] ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 .mu.M Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E .cntdot. ATP .cntdot. Ca2 is .apprx. 2.2 .times. 10-10 M2 (K0.5 = 15 .mu.M). The rate constant for the slow phase of the biphasic reaction of E .apprx. P .cntdot. Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 .mu.M to 10 nM, with half-maximal increase at 1.7 .mu.M Ca2+. This shows that Ca2+ is dissociating from a different species, aE .cntdot. ATP .cntdot. Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k .ltoreq. 45 s-1. It is concluded that steady-state turnover of the ATPase under most conditions occurs through the E .cntdot. ATP .cntdot. Ca2 pathway, which has a relatively low affinity for Ca2+, not the pathway through cE .cntdot. Ca2 (or "E1 .cntdot. Ca2"). This results in 11-17% unphosphorylated enzyme in the steady state at saturing [ATP] and [Ca2+] because the kb step is partly rate limiting. The two pathways for phosphorylation can result in nonlinear Lineweaver-Burk plots for ATP and initial overshoots of phosphoenzyme levels.

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