title: Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon.
abstract: Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.
authors.affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Patterson
authors.forename: Diana
authors.initials: D
authors.lastname: Vineyard
authors.forename: Jennifer
authors.initials: J
authors.lastname: Thomas-Wohlever
authors.forename: Ramona
authors.initials: R
authors.lastname: Behshad
authors.forename: Morris
authors.initials: M
authors.lastname: Burke
authors.forename: Irene
authors.initials: I
authors.lastname: Lee
mesh_terms: D049069:ATP-Dependent Proteases; D000225:Adenine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D049070:Protease La; D011487:Protein Conformation; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D009711:Nucleotides; D010446:Peptide Fragments; D010455:Peptides; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D014357:Trypsin; C483567:Lon protein, E coli; D049070:Protease La; D043583:Nucleoside-Triphosphatase; D000225:Adenine