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

Abstract

A group of mutant uncA alleles, affecting essential residues of the n-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 F,-ATPase.The amino acid substitution found was Glu-299 4 Lys, which is predicted to lie in an a-helix in a-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 4 Phe.Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F,ATPase were characterized as follows: uncA401, Ser-373 + Phe; uncA447, Gly-351-Asp; uncA453, Ser-375 4 Phe.We predict here that the nucleotide-binding domain of a-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 a-subunit, at the C-terminal end of the predicted nucleotide-binding domain.This segment is suggested to be important for &X-@ intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.' The abbreviation used is: kb, kilobase pairs.