Inactivation and phosphorylation of sarcoplasmic reticulum Ca 2+-ATPase by Mg · ATP analogues Rh(III)-ATP and Co(III)-ATP

Abstract

The interactions of sarcoplasmic reticulum Ca 2+-ATPase with the Mg · ATP analogues Rh(H 2O) 4ATP and Co(NH 3) 4ATP have been examined. Co(NH 3) 4ATP slowly inactivates Ca 2+-ATPase in a first order process, with a rate constant of 1.13 × 10 −3 s −1 and an apparent inactivation constant, K I , of 32 m m. Rh(H 2O) 4ATP likewise inactivates sarcoplasmic reticulum Ca 2+-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H 2O) 4ATP] is biphasic. The x-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca 2+-ATPase by Rh(H 2O) 4ATP of K I1 = 30 μM and K I2 = 221 μM. The corresponding values of k 2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 × 10 −4 s −1. Tridentate Rh(H 2O) 3ATP also inhibits Ca 2+-ATPase, but only after much longer incubation times. Ca 2+-ATPase inactivation is accompanied by incorporation of radioactivity from γ- 32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca 2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca 2+-dependent ATPase activity in this experiment was 2.19 × 10 −4 s −1, and the first-order rate constant for Ca 2+-dependent E-P formation was 2.07 × 10 −4 s −1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca 2+-ATPase by Rh(H 2O) 4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca 2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H 2O) 4ATP is binding to the catalytic nucleotide site on Ca 2+-ATPase and producing a highly stable, phosphorylated intermediate.