Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate.
The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D006180:Proton-Translocating ATPases; D014316:Tritium
D010761:Phosphorus Radioisotopes; D014316:Tritium; C032197:beta-aspartyl phosphate; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases