Reaction mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. ATP hydrolysis with CaATP as a substrate and role of divalent cation.
ATP hydrolysis with CaATP as a substrate was characterized at 0 degrees C and pH 7.0 using purified ATPase preparations of sarcoplasmic reticulum and compared with that with MgATP as a substrate. The maximal rate of enzyme phosphorylation and the Km value for the phosphorylation were 8 to 10 times less for CaATP than for MgATP. Each substrate appeared to act as a competitive inhibitor with respect to the other in enzyme phosphorylation. The phosphoenzyme formed from CaATP turned over slowly because the conversion rate of the ADP-sensitive (E1P) to ADP-insensitive (E2P) phosphoenzyme was very slow. E2Ps, formed from both CaATP and MgATP, were similar in that KCl, MgCl2, or ATP accelerated their decomposition. Their sensitivity to KCl and/or ATP was retained even after a long incubation with excess EDTA. When the enzyme had been phosphorylated from CaATP, calcium remained bound to the enzyme even in the presence of excess EDTA. The observed parallelism between the amount and behavior of the enzyme-bound calcium and those of E2P strongly suggests that 1 mol of E2P has 1 mol of tightly bound calcium. During steady state ATP hydrolysis with CaATP as a substrate, a significant amount of the enzyme-ATP complex accumulated as a reaction intermediate because of slow dissociation of CaATP from the CaATP-enzyme complex and slow enzyme phosphorylation from the CaATP-enzyme complex. These results indicate that Mg2+ is not essential for the turnover of the calcium pump ATPase. It was proposed that the metal component of the substrate basically determines affinity of the substrate to the enzyme and the catalytic mechanism of subsequent reaction steps.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D010761:Phosphorus Radioisotopes; D015636:Magnesium Chloride; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002122:Calcium Chloride; D002118:Calcium