Inhibition of the adenosinetriphosphatase activity of Escherichia coli F1 by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is due to modification of several carboxyls in the beta subunit.
Reaction of the ATPase of Escherichia coli (ECF1) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) resulted in a time- and concentration-dependent inhibition of ATPase activity. The inactivation was greatly reduced by Mg2+ ions. Close to 13 mol of EDC per mol of ECF1 was incorporated into the enzyme at 95% inhibition of ATPase activity. Two-thirds of the label was found to be associated with subunit beta with a stoichiometry of about 3 mol of EDC per mol of beta. Cleavage of EDC-modified subunit beta with cyanogen bromide and fractionation of the peptides by high-pressure liquid chromatography revealed a short segment of 33 amino acids (CB8, residues 162-194) containing 3 mol of EDC per mol of peptide. In tryptic peptide maps, two EDC-labeled fragments could be identified (T18, residues 166-183, and T20, residues 186-202). The analyses were complicated by significant internal cross-linking within the beta subunit induced by EDC. The results show that EDC modifies multiple sites in a short segment of subunit beta which includes the glutamic acids modified by dicyclohexylcarbodiimide in F1 from both E. coli and PS3. In addition to covalent modification, EDC also promoted the formation of intersubunit cross-links. The predominant cross-linked product was identified as a beta-epsilon complex by antibody binding experiments.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D002234:Carbodiimides; D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases; D005022:Ethyldimethylaminopropyl Carbodiimide