Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase.
The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain. A recent proposal (Maggio, M. B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A. E. (1987) J. Biol. Chem. 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit. Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide. In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu. The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu). Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants. The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding. The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005838:Genotype; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
D046911:Macromolecular Substances; D009711:Nucleotides; D006180:Proton-Translocating ATPases; D008239:Lysine