Sarcoplasmic reticulum adenosinetriphosphatase phosphorylation from inorganic phosphate. Theoretical and experimental reinvestigation.
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, we measured medium Pi oxygen exchange, [32P]-phosphoenzyme formation, and intrinsic fluorescence. We found that contrarily to the usual assumption the substrate binding step in the phosphorylation direction at pH 6.0, KCl = 0, and 23 degrees C is a slow process whose bimolecular rate constant is around 5 X 10(3) M-1 s-1 for both Mg and Pi binding. We confirm [Lacapère, J. J., Gingold, M. P., Champeil, P., & Guillain, F. (1981) J. Biol. Chem. 256, 2302-2306] that, in a second step, the establishment of a covalent bond between the bound Pi and the enzyme is formed with a rate constant greater than or equal to 20 s-1 whereas the dephosphorylation rate constant is 2-3 s-1. These results imply that 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 addition is mainly due to the augmentation of the hydrolysis rate constant. In addition we propose that the strong inhibition by large amounts of MgCl2 is due to the formation of an E? . Mg complex unfit for phosphorylation by Pi.(ABSTRACT TRUNCATED AT 250 WORDS)
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008433:Mathematics; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D015636:Magnesium Chloride; D011189:Potassium Chloride; D000251:Adenosine Triphosphatases; D008274:Magnesium