title: Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli.
abstract: Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
authors.forename: M D
authors.initials: MD
authors.lastname: Adams
authors.forename: D J
authors.initials: DJ
authors.lastname: Maguire
authors.forename: D L
authors.initials: DL
authors.lastname: Oxender
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007930:Leucine