Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.
The carboxyl group activating reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) interacts with the Neurospora plasma membrane H+-ATPase in at least three different ways. This reagent irreversibly inhibits ATP hydrolysis with kinetics that are pseudo-first-order at several concentrations of EEDQ, and an appropriate transform of these data suggests that 1 mol of EEDQ inactivates 1 mol of the H+-ATPase. Inhibition probably involves activation of an ATPase carboxyl group followed by a nucleophilic attack by a vicinal nucleophilic functional group in the ATPase polypeptide chain, resulting in an intramolecular cross-link. The enzyme is protected against EEDQ inhibition by MgATP in the presence of vanadate, a combination of ligands that has previously been shown to "lock" the H+-ATPase in a conformation that presumably resembles the transition states of the enzyme phosphorylation and dephosphorylation reactions, but is not protected by the substrate analogue MgADP, which is consistent with the notion that one or both of the residues involved in the EEDQ-dependent inhibitory intramolecular cross-linking reaction normally participate in the transfer of the gamma-phosphoryl group of ATP, or are near those that do. The ATPase is also labeled by the exogenous nucleophile [14C]glycine ethyl ester in an EEDQ-dependent reaction, and the labeling is diminished in the presence of MgATP plus vanadate. However, peptide maps of [14C]glycine ethyl ester labeled ATPase demonstrate that the labeling is not related to the EEDQ inhibition reaction in any simple way.(ABSTRACT TRUNCATED AT 250 WORDS)
D002462:Cell Membrane / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011804:Quinolines; D013861:Thiocyanates; C000470:EEDQ; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate