The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly.

Abstract

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.