Phosphoenzyme formation from ATP in the ATPase of sarcoplasmic reticulum. Effect of KCl or ATP and slow dissociation of ATP from precursor enzyme-ATP complex.
The ATP-dependent phosphoenzyme formation and its reversal were studied at 0 degrees C and pH 7.0 in the ATPase of sarcoplasmic reticulum. Addition of KCl or several other salts (approximately 100 mM) decreased the maximum rate of ADP-induced dephosphorylation of phosphoenzyme as well as the apparent affinity of the phosphoenzyme toward ADP. High ATP had a similar effect on the latter, whereas it had little effect on the former. In contrast, high KCl or a considerable change in the ionic strength had little effect on the initial rate of phosphoenzyme formation at saturating ATP concentrations. During steady state phosphorylation at 1.0 mM MgCl2 and 5.0 mM CaCl2 in the absence of added KCl, a significant amount of [gamma-32P]ATP remained bound to the enzyme even when the enzyme concentration was much in excess over that of [gamma-32P]ATP. Evidence is presented that this enzyme-ATP complex represents a precursor to the phosphoenzyme. ATP dissociated slowly (0.20 s-1) from this enzyme-ATP complex and addition of high KCl or other salts accelerated its dissociation. In contrast, when the enzyme was complexed with adenyl-5'-yl (beta, gamma-methylene)diphosphonate in the absence of added KCl under these conditions, dissociation of the nucleotide from the complex as estimated in the displacement experiment with [gamma-32P]ATP, was found to be much faster than that of ATP.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D010750:Phosphoproteins; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases