Rat liver mitochondrial ATP synthase. Effects of mutations in the glycine-rich region of a beta subunit peptide on its interaction with adenine nucleotides

J Biol Chem. 1990 Aug 25;265(24):14632-7.


The beta subunit of the rat liver mitochondrial ATP synthase contains a glycine-rich amino acid sequence implicated in binding nucleotides by its similarity to a sequence found in many other nucleotide-binding proteins. A C-terminal three-quarter-length rat liver beta subunit fragment (Glu122 through Ser479), containing this homology region, interacts with adenine nucleotides (Garboczi, D.N., Hullihen, J.H., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 15694-15698). Here we directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion. Twenty-one mutations within the glycine-rich region of the beta subunit cDNA were randomly generated. Wild-type and mutant beta subunit proteins were purified from overexpressing Escherichia coli strains. The mutant proteins were screened for changes in their interaction with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), a fluorescent nucleotide analog. Only one mutant protein bearing two amino acid changes (Gly153----Val, Gly156----Arg) exhibited a fluorescence enhancement higher than that of the wild-type "control." Further analysis of this protein revealed a lower affinity for TNP-ATP (Kd = 10 microM) compared with wild type (Kd = 5 microM). In addition, a mutant from which amino acids Gly149-Lys214 had been deleted was prepared. This mutant protein, which lacks the entire glycine-rich region, also displayed a marked reduction in affinity for TNP-ATP (Kd greater than 60 microM). Prior addition of 0.5 mM ATP significantly reduced the binding of TNP-ATP to both the double and deletion mutants. The altered interaction of nucleotide with both glycine-rich region mutants points to the involvement of this region in the binding site. Further, this work shows that a beta subunit protein that lacks the glycine-rich homology region can still interact with nucleotide, indicating that one or more additional regions of this subunit contribute to the nucleotide binding site.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA / genetics
  • Escherichia coli / genetics
  • Fluorescent Dyes
  • Glycine*
  • Kinetics
  • Macromolecular Substances
  • Mitochondria, Liver / enzymology*
  • Mitochondria, Liver / metabolism
  • Molecular Sequence Data
  • Mutation*
  • Plasmids
  • Proton-Translocating ATPases / genetics*
  • Rats
  • Recombinant Proteins / metabolism


  • Fluorescent Dyes
  • Macromolecular Substances
  • Recombinant Proteins
  • 2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate
  • Adenosine Triphosphate
  • DNA
  • Proton-Translocating ATPases
  • Glycine