Dynamic reversal of enzyme carboxyl group phosphorylation as the basis of the oxygen exchange catalyzed by sarcoplasmic reticulum adenosine triphosphatase

Abstract

Millisecond mixing and quenching experiments demonstrate an apparent t1/2 for the labeling of phosphorylated sarcoplasmic reticulum ATPase by 32Pi at pH 6 and 30 degrees C of 30 to 40 ms. Under the same conditions, the rate of exchange of water oxygens with inorganic phosphate (Pi) is about 40 mol of H2O exchanged with Pi per 10(6) g of protein per s. Theoretical equations are developed for the expected 32P-labeling pattern given various comparative rates of flux between Pi and the Michaelis complex and between the Michaelis complex and phosphorylated enzyme. The results show that the rapid reversal of the formation of the phosphorylated enzyme is a major source of the oxygen exchange and are consistent with such reversal being the only source.