Sequential hydrolysis of the gamma- and beta-phosphate groups of ATP by the ATP diphosphohydrolase from pig pancreas.

The ATP diphosphohydrolase (EC 3.6.1.5) from pig pancreas hydrolyzes triphospho- and diphosphonucleosides. The reaction products of ATP hydrolysis are ADP, AMP and orthophosphate, but AMP accumulates at a faster rate than ADP. A time-course study showed a simultaneous breakdown of ATP and ADP with initial rates for ATP and ADP hydrolysis of 2.1 and 3.8 mumol/min per mg protein, respectively. However, the rates reached similar values toward the end of the incubation period. According to double reciprocal plots and Dixon plots, the Km values for ATP and ADP are similar, Vmax for ADP hydrolysis is twice the Vmax for ATP hydrolysis and both nucleotides are competitive inhibitors of the other with their Ki values similar to their Km. These results are consistent with a sequential hydrolysis of the two diphosphoester bonds of ATP: ATP first binds to the enzyme, its gamma-phosphate group is hydrolyzed and released, resulting in an enzyme-ADP complex which either breaks down to free enzyme and ADP or is further processed via hydrolysis of the beta-phosphate group, releasing free enzyme, AMP and Pi. The experimental data showed that the processing step is favored.