Evidence for an aspartyl phosphate residue at the active site of sodium and potassium ion transport adenosine triphosphatase.

In order to characterize part of an active center of sodium and potassium ion transport adenosine triphosphatase, namely the active site of phosphorylation, the enzyme was phosphorylated with [32P]ATP and denatured with acid. The denatured [3SP]phosphoenzyme was digested to a limit [32P]phosphotripeptide with Pronase. The limit phosphotripeptide was compared with the synthetic model compounds, [3?P]prolylphosphoaspartyllysine and [32P]prolylphosphoglutamyllysine with respect to pH-hydrolysis profile, sensitivity to digestion by carboxypeptidase B, and isoelectric point. The active site phosphotripeptide closely resembled the synthetic phosphoaspartyl peptide and clearly differed from the synthetic phosphoglutamyl peptide in these respects. These similarities are strong, but not conclusive, evidence that the active site of this enzyme is the /3-carboxyl group of an aspartic acid residue. Previous work has shown that the limit phosphotripeptide contains an NH2-terminal serine or threonine residue and a COOH-terminal lysine residue. The enzyme source was guinea pig kidneys. In order to characterize the active site before denaturation, the sensitivity of the phosphate group to hydroxylamine was tested. The enzyme was first treated with N-ethylmaleimide to inhibit acceleration of dephosphorylation due to a resemblance between hydroxylamine and potassium ion. The treated enzyme was subsequently phosphorylated by [32P]adenosine triphosphate and the phosphoenzyme was chased with unlabeled adenosine triphosphate. During the chase, addition of hydroxylamine produced a dephosphorylation which was 45-fold faster than that of the isolated phosphotripeptide under the same experimental conditions. It is concluded that the phosphoenzyme has sufficient sensitivity to hydroxylamine before denaturation to qualify as an acyl phosphate.