Sequential reactions in Pi utilization for ATP synthesis by sarcoplasmic reticulum.

Abstract

Incorporation of Pi and ATP synthesis by sarcoplasmic reticulum ATPase were studied by rapid quench methods in order to demonstrate whether protein phosphorylation obtained in various experimental conditions leads to intermediates of the same reaction chain, or to products of independent reactions. Phosphorylation occurs rapidly (k = 30 s-‘) when saturating Pi is added to vesicles preincubated with ethylene glycol bis(P-aminoethyl ether)iV,iV,iV’,iV’-tetraacetic acid (EGTA), while it is much slower (k = 2.0 s-‘) in vesicles loaded actively (with calcium in the presence of ATP) previous to sequential addition of EGTA and Pi. If the reaction is started by simultaneous rather than sequential addition of EGTA and Pi, a lag period is observed and phosphorylation occurs with identical kinetics in loaded and nonloaded vesicles. In all cases, independent of the different kinetics of ATPase phosphorylation with Pi, decay of the resulting phosphoenzyme occurs with identical kinetics upon Pi dilution, consistent with hydrolytic cleavage of the same species. The rate constant for the hydrolytic reaction (8 to 11 s-l) is equal to the turnover of the enzyme operating with ATP as the substrate. Addition of Pi and ADP to loaded vesicles, independent of whether EGTA is added previous to or simultaneous with Pi, results in ATP synthesis with kinetics similar to those of Pi incorporation into the enzyme. It is concluded that ATPase phosphorylation with Pi occurs exclusively when the enzyme resides in specific reactive states which are part of the same catalytic cycle. Observed differences in the kinetics of phosphorylation are related to different states in which the enzyme resides in various experimental conditions and reflect conversion of the enzyme to a reactive state. Independent of the kinetics of ATPase phosphorylation with Pi, the resulting phosphoenzyme acquires the ability to synthesize ATP in the presence of high intravesicular Ca’+.