Sarcoplasmic reticulum adenosine triphosphatase phosphorylation from inorganic phosphate. Theoretical and experimental reinvestigation

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Abstract
Pi phosphorylation of sarcoplasmic reticulum (SR) vesicles in the absence of Ca was reinvestigated. Theoretical analysis shows that, for various substrate concentrations, the time dependence of phosphoenzyme formation does not allow determination of an unambiguous reaction scheme or estimation of the stoichiometry of the reaction. To overcome this difficulty, medium Pi oxygen exchanges, [32P]-phosphoenzyme formation and intrinsic fluorescence were measured. Contrary to the usual assumption the substrate binding step in the phosphorylation direction at pH 6.0, KCl = 0 and 23.degree. C is a slow process whose bimolecular rate constant is around 5 .times. 103 M-1 s-1 for both Mg and Pi binding. It was confirmed that in a second step, the establishment of a covalent bond between the bound Pi and the enzyme is formed with a rate constant .gtoreq. 20 s-1 whereas the dephosphorylation rate constant is 2-3 s-1. Under optimal conditions for phosphorylation, the enzyme is almost entirely phosphorylated at concentrations of 20 mM MgCl2 and 20 mM Pi. Study of the phosphorylation reaction under various experimental conditions shows that reduction of the phosphoenzyme level upon KCl additions is mainly due to the augmentation of the hydrolysis rate constant. The strong inhibition by large amounts of MgCl2 is probably due to the formation of an E?.cntdot.Mg complex unfit for phosphorylation by Pi. Diminution of the phosphoenzyme level when the pH increases reflects higher enzyme sensitivity to Mg inhibition at alkaline pH. Results for inhibition by ATP of the phosphorylation reaction at pH 6.0 and KCl = 0 are also presented and discussed in the light of the data available in the literature.

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