J. Biol. Chem., Vol. 263, Issue 18, 8727-8734, Jun, 1988

Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits

BJ Hwang, KM Woo, AL Goldberg and CH Chung
Department of Zoology, College of Natural Science, Seoul National University, Korea.

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.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				X. Dong, M. Kato-Murayama, T. Muramatsu, H. Mori, M. Shirouzu, Y. Bessho, and S. Yokoyama The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli Protein Sci., March 1, 2007; 16(3): 528 - 534. [Abstract] [Full Text] [PDF]

				B. M. Burton and T. A. Baker Remodeling protein complexes: Insights from the AAA+ unfoldase ClpX and Mu transposase Protein Sci., August 1, 2005; 14(8): 1945 - 1954. [Abstract] [Full Text] [PDF]

				F. Moussaoui, F. Vangroenweghe, K. Haddadi, Y. Le Roux, F. Laurent, L. Duchateau, and C. Burvenich Proteolysis in Milk During Experimental Escherichia coli Mastitis J Dairy Sci, September 1, 2004; 87(9): 2923 - 2931. [Abstract] [Full Text] [PDF]

				J. M. Bateman, M. Iacovino, P. S. Perlman, and R. A. Butow Mitochondrial DNA Instability Mutants of the Bifunctional Protein Ilv5p Have Altered Organization in Mitochondria and Are Targeted for Degradation by Hsp78 and the Pim1p Protease J. Biol. Chem., November 27, 2002; 277(49): 47946 - 47953. [Abstract] [Full Text] [PDF]

				J. H. Lo, T. A. Baker, and R. T. Sauer Characterization of the N-terminal repeat domain of Escherichia coli ClpA--A class I Clp/HSP100 ATPase Protein Sci., March 1, 2001; 10(3): 551 - 559. [Abstract] [Full Text]

				E. C. Schirmer, D. M. Ware, C. Queitsch, A. S. Kowal, and S. L. Lindquist Subunit interactions influence the biochemical and biological properties of Hsp104 PNAS, January 23, 2001; (2001) 31568098. [Abstract] [Full Text]

				C. Li, Y. P. Tao, and L. D. Simon Expression of Different-Size Transcripts from the clpP-clpX Operon of Escherichia coli during Carbon Deprivation J. Bacteriol., December 1, 2000; 182(23): 6630 - 6637. [Abstract] [Full Text]

				E. Ekaza, L. Guilloteau, J. Teyssier, J.-P. Liautard, and S. Köhler Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice Microbiology, July 1, 2000; 146(7): 1605 - 1616. [Abstract] [Full Text]

				S. Krause, W. Pansegrau, R. Lurz, F. de la Cruz, and E. Lanka Enzymology of Type IV Macromolecule Secretion Systems: the Conjugative Transfer Regions of Plasmids RP4 and R388 and the cag Pathogenicity Island of Helicobacter pylori Encode Structurally and Functionally Related Nucleoside Triphosphate Hydrolases J. Bacteriol., May 15, 2000; 182(10): 2761 - 2770. [Abstract] [Full Text]

				H. L. Wilson, M. S. Ou, H. C. Aldrich, and J. Maupin-Furlow Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome J. Bacteriol., March 15, 2000; 182(6): 1680 - 1692. [Abstract] [Full Text]

				O. Kandror, M. Sherman, and A. Goldberg Rapid Degradation of an Abnormal Protein in Escherichia coli Proceeds through Repeated Cycles of Association with GroEL J. Biol. Chem., December 31, 1999; 274(53): 37743 - 37749. [Abstract] [Full Text] [PDF]

				A. L. Horwich, E. U. Weber-Ban, and D. Finley Chaperone rings in protein folding and degradation PNAS, September 28, 1999; 96(20): 11033 - 11040. [Abstract] [Full Text] [PDF]

				M. Pak, J. R. Hoskins, S. K. Singh, M. R. Maurizi, and S. Wickner Concurrent Chaperone and Protease Activities of ClpAP and the Requirement for the N-terminal ClpA ATP Binding Site for Chaperone Activity J. Biol. Chem., July 2, 1999; 274(27): 19316 - 19322. [Abstract] [Full Text] [PDF]

				S. Santagata, D. Bhattacharyya, F.-H. Wang, N. Singha, A. Hodtsev, and E. Spanopoulou Molecular Cloning and Characterization of a Mouse Homolog of Bacterial ClpX, a Novel Mammalian Class II Member of the Hsp100/Clp Chaperone Family J. Biol. Chem., June 4, 1999; 274(23): 16311 - 16319. [Abstract] [Full Text] [PDF]

				L. Van Melderen and S. Gottesman Substrate sequestration by a proteolytically inactive Lon mutant PNAS, May 25, 1999; 96(11): 6064 - 6071. [Abstract] [Full Text] [PDF]

				J. R. Hoskins, M. Pak, M. R. Maurizi, and S. Wickner The role of the ClpA chaperone in proteolysis by ClpAP PNAS, October 13, 1998; 95(21): 12135 - 12140. [Abstract] [Full Text] [PDF]

				S. J. Yoo, H. H. Kim, D. H. Shin, C. S. Lee, I. S. Seong, J. H. Seol, N. Shimbara, K. Tanaka, and C. H. Chung Effects of the Cys Mutations on Structure and Function of the ATP-dependent HslVU Protease in Escherichia coli. THE CYS287 TO VAL MUTATION IN HslU UNCOUPLES THE ATP-DEPENDENT PROTEOLYSIS BY HslVU FROM ATP HYDROLYSIS J. Biol. Chem., September 4, 1998; 273(36): 22929 - 22935. [Abstract] [Full Text] [PDF]

				E. C. Schirmer, C. Queitsch, A. S. Kowal, D. A. Parsell, and S. Lindquist The ATPase Activity of Hsp104, Effects of Environmental Conditions and Mutations J. Biol. Chem., June 19, 1998; 273(25): 15546 - 15552. [Abstract] [Full Text] [PDF]

				R. Grimaud, M. Kessel, F. Beuron, A. C. Steven, and M. R. Maurizi Enzymatic and Structural Similarities between the Escherichia coli ATP-dependent Proteases, ClpXP and ClpAP J. Biol. Chem., May 15, 1998; 273(20): 12476 - 12481. [Abstract] [Full Text] [PDF]

				J. M. Jones, D. J. Welty, and H. Nakai Versatile Action of Escherichia coli ClpXP as Protease or Molecular Chaperone for Bacteriophage Mu Transposition J. Biol. Chem., January 2, 1998; 273(1): 459 - 465. [Abstract] [Full Text] [PDF]

				I. E. Ichetovkin, G. Abramochkin, and T. E. Shrader Substrate Recognition by the Leucyl/Phenylalanyl-tRNA-protein Transferase. CONSERVATION WITHIN THE ENZYME FAMILY AND LOCALIZATION TO THE TRYPSIN-RESISTANT DOMAIN J. Biol. Chem., December 26, 1997; 272(52): 33009 - 33014. [Abstract] [Full Text] [PDF]

				E. C. Schirmer and S. Lindquist Interactions of the chaperone Hsp104 with yeast Sup35 and mammalian PrP PNAS, December 9, 1997; 94(25): 13932 - 13937. [Abstract] [Full Text] [PDF]

				I Levchenko, M Yamauchi, and T A Baker ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes & Dev., June 15, 1997; 11(12): 1561 - 1572. [Abstract] [PDF]

				L. Hoffman and M. Rechsteiner Nucleotidase Activities of the 26S Proteasome and Its Regulatory Complex J. Biol. Chem., December 20, 1996; 271(51): 32538 - 32545. [Abstract] [Full Text] [PDF]

				G. Abramochkin and T. E. Shrader Aminoacyl-tRNA Recognition by the Leucyl/Phenylalanyl-tRNA-Protein Transferase J. Biol. Chem., September 13, 1996; 271(37): 22901 - 22907. [Abstract] [Full Text] [PDF]

				S. J. Yoo, J. H. Seol, D. H. Shin, M. Rohrwild, M.-S. Kang, K. Tanaka, A. L. Goldberg, and C. H. Chung Purification and Characterization of the Heat Shock Proteins HslV and HslU That Form a New ATP-dependent Protease in Escherichia coli J. Biol. Chem., June 14, 1996; 271(24): 14035 - 14040. [Abstract] [Full Text] [PDF]

				I Levchenko, L Luo, and T A Baker Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes & Dev., October 1, 1995; 9(19): 2399 - 2408. [Abstract] [PDF]

				G. Abramochkin and T. E. Shrader The Leucyl/Phenylalanyl-tRNA-Protein Transferase J. Biol. Chem., September 1, 1995; 270(35): 20621 - 20628. [Abstract] [Full Text] [PDF]

				J. H. Seol, S. H. Baek, M.-S. Kang, D. B. Ha, and C. H. Chung Distinctive Roles of the Two ATP-binding Sites in ClpA, the ATPase Component of Protease Ti in Escherichia coli J. Biol. Chem., April 7, 1995; 270(14): 8087 - 8092. [Abstract] [Full Text] [PDF]

				C. Chung Proteases in Escherichia coli Science, October 15, 1993; 262(5132): 372 - 374. [PDF]

				E. C. Schirmer, D. M. Ware, C. Queitsch, A. S. Kowal, and S. L. Lindquist Subunit interactions influence the biochemical and biological properties of Hsp104 PNAS, January 30, 2001; 98(3): 914 - 919. [Abstract] [Full Text] [PDF]