Identification of Amino‐Acid Substitutions in the Proteolipid Subunit of the ATP Synthase from Dicyclohexylcarbodiimide‐Resistant Mutants of Escherichia coli

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Abstract
The amino acid sequence of the proteolipid subunit of the ATP synthase was analyzed in 6 mutant strains from E. coli K12, selected for their increased resistance towards the inhibitor N,N''-dicyclohexylcarbodiimide. All 6 inhibitor-resistant mutants were altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I, this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed. These 2 amino acid substitutions did not influence functional properties of the ATPase complex. ATPase and ATP-dependent proton-translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexylcarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type and thereby inhibited the activity of the ATPase complex. The residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these 2 residues are in close vicinity and would thus provide a 1st hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these 2 residues separated by a segment of 32 residues.

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