Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments

• Published in: Acta biochimica polonica Vol. 55; no. 2; pp. 281 - 296
• Main Authors: Melnikov, Edward E, Andrianova, Anna G, Morozkin, Andrey D, Stepnov, Anton A, Makhovskaya, Oksana V, Botos, Istvan, Gustchina, Alla, Wlodawer, Alexander, Rotanova, Tatyana V
• Format: Journal Article
• Language: English
• Published: Poland 01.01.2008
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Catalytic Domain; Chymotrypsin; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Peptide Hydrolases; Protease La - chemistry; Protease La - genetics; Protease La - metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 0001-527X; 1734-154X
• DOI: 10.18388/abp.2008_3075

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.