Source of the Oxygen in the C-O-P Linkage of the Acyl Phosphate in Transport Adenosine Triphosphatases

The source of the oxygen in the C-O-P linkage of the acyl phosphate in the Na+, K+-ATPase of porcine kidney and the Ca2+, Mg2+-ATPase of rabbit muscle sarcoplasmic reticulum has been assessed. Formation of the Na+, K+-ATPase acyl phosphate in presence of ouabain and [18O]Pi results in the C-O-P bridge oxygen remaining nonisotopic. Thus, the mode of entry of Pi into the acyl phosphate is by attack of a carboxylate oxygen on the phosphorus atom and displacement of a hydroxyl group. When the acyl phosphate of the Ca2+, Mg2+-ATPase or the Na+, K+-ATPase is formed in the presence of H18OH, the C-O-P bridge oxygen also remains nonisotopic. These results show that the acyl phosphate is cleaved by water oxygen attack on the phosphorus atom and rules out any acyl transfer to an enzyme group and subsequent hydrolysis by attack of water oxygen on the acyl carbon atom. The phosphoryl oxygens of the Na+,K+-ATPase phosphoenzyme at 15° derived either from [18O]Pi in the presence of ouabain or from ATP in the presence of H18OH were observed to undergo exchange with medium water, not accounted for by exchange of Pi oxygens with HOH and incorporation of the Pi into the protein-bound acyl phosphate.