title: ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli.
abstract: Protein degradation in Escherichia coli involves the ATP-dependent serine protease La. Protease La is a homotetramer with one proteolytic and one ATP binding site per monomer. Its proteolytic activity has been shown to be highly increased by simultaneous hydrolysis of ATP, which is essential for the degradation of protein substrates by this enzyme. We have cloned and purified a proteolytically inactive La mutant, in which the catalytically active serine residue at position 679 was replaced by alanine. Fluorescence and circular dichroism spectra of the purified wild type and mutant enzyme revealed identical conformations of the proteins. Based on this observation, the catalytic properties of the wild type enzyme and the S679A mutant were compared. Although the S679A mutant lacks proteolytic activity toward both peptide and protein substrates under all conditions investigated, its ATPase activity is completely unaffected by the removal of the protease activity. Since protein substrates stimulate the ATP-dependent hydrolysis of peptides by protease La, it has been argued that this stimulation is due to interactions with a regulatory binding site on the enzyme. In accordance with this model, protein substrates such as alpha-casein and denatured bovine serum albumin stimulate the ATPase activity of the S679A mutant to the same degree as in the active protease. Therefore, the intrinsic ATPase activity of protease La as well as its stimulation is not dependent on the simultaneous hydrolysis of the protein substrate.
authors.affiliation: Institut für Biohysik und Physikalische Biochemie, Universität Regensburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Fischer
authors.forename: R
authors.initials: R
authors.lastname: Glockshuber
mesh_terms: D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D006360:Heat-Shock Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases