On the mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. Occurrence of two types of phosphoenzyme intermediates in the presence of KCl.
The steady state kinetics of ATP hydrolysis by partially purified adenosine triphosphatase preparations of sarcoplasmic reticulum was investigated at 0 degrees C and pH 7.0 in 2.0 mM MgCl2, 20 microM [gamma-32P]ATP, 20 microM CaCl2, and various concentrations of KCl in the presence and absence of 12% dimethyl sulfoxide. The steady state phosphoenzyme formed under these conditions could be resolved kinetically into ADP-sensitive and ADP-insensitive forms. These steady state kinetic data were analyzed according to a scheme in which the ADP-sensitive and ADP-insensitive phosphoenzymes occur sequentially, and Pi is derived from the latter. The KCl-dependent turnover rate of the ADP-insensitive phosphoenzyme that was estimated according to this scheme was in good agreement with the directly measured hydrolysis rate constant of the ADP-insensitive phosphoenzyme. In addition, the time course of the decomposition of the total amount of phosphoenzyme, measured after a steady state level was reached in 20 mM KCl and further phosphorylation was prevented by addition of excess ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, was also in agreement with that calculated according to this scheme using values of the rate constants estimated from the amounts of the ADP-sensitive and ADP-insensitive phosphoenzymes and the rate of ATP hydrolysis. These results, together with our previous findings, support the view that this scheme describes the mechanism of ATP hydrolysis in the presence of KCl.
D000255:Adenosine Triphosphate; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases