title: Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits.
abstract: In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti. This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis. Both have been purified to homogeneity by conventional procedures using [3H]casein as the substrate. The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration. Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with [3H] diisopropyl fluorophosphate. It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits. The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably. The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins. Component A also shows ATPase activity which can be stimulated by casein. Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein. Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP. Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis. Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP. Casein increases the Vmax for ATP without affecting the Km. A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis. Ca2+ and Mn2+ partially support these activities. Thus, protease Ti shares many unusual properties with protease La (e.g. coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.
authors.affiliation: Department of Zoology, College of Natural Science, Seoul National University, Korea.
authors.forename: B J
authors.initials: BJ
authors.lastname: Hwang
authors.forename: K M
authors.initials: KM
authors.lastname: Woo
authors.forename: A L
authors.initials: AL
authors.lastname: Goldberg
authors.forename: C H
authors.initials: CH
authors.lastname: Chung
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases