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Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities
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SYSNO ASEP 0436201 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities Author(s) Ambro, L. (SK)
Pevala, V. (SK)
Ondrovičová, G. (SK)
Bellová, J. (SK)
Kunová, N. (SK)
Kutejová, Eva (MBU-M) RID
Bauer, J. (SK)Source Title FEBS Journal - ISSN 1742-464X
Roč. 281, č. 7 (2014), s. 1784-1797Number of pages 14 s. Language eng - English Country GB - United Kingdom Keywords ATP-dependent protease ; glycine loop ; human Lon protease Subject RIV CE - Biochemistry Institutional support MBU-M - RVO:61388971 UT WOS 000333676000007 DOI 10.1111/febs.12740 Annotation Lon, also called protease La, is an ATP-dependent protease present in all kingdoms of life. It is involved in protein quality control and several regulatory processes. Eukaryotic Lon possesses three domains, an N-terminal domain, an ATPase domain and a proteolytic domain. It requires ATP hydrolysis to digest larger, intact proteins, but can cleave small, fluorogenic peptides such as Glu-Ala-Ala-Phe-MNA by only binding, but not hydrolyzing, ATP. Both ATPase and peptidase activities can be stimulated by the binding of a larger protein substrate, such as -casein. To better understand its mechanism of action, we have prepared several point mutants of four conserved residues of human Lon (G893A, G893P, G894A, G894P, G894S, G893A-G894A, G893P-G894A, G893A-G894P, T880V, W770A, W770P) and studied their ATPase, protease and peptidase activities. Our results show that mutations to Gly894 enhance its basal ATPase activity but do not change its -casein-stimulated activity. The loop containing Gly893 and Gly894, which flanks Lon's proteolytic active site, therefore appears to be involved in the conformational change that occurs upon substrate binding. Furthermore, mutations to Trp770 have the same general effects on the ATPase activity as mutations to Gly893, indicating that Trp770 is involved in ATPase stimulation. We have also established that this loop does not need to move in order to cleave small, fluorogenic peptides, but does move during the digestion of -casein. Finally, we also noted that Lon's ability to digest small peptides can be inhibited by moderate ATP concentrations. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2015
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