Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities
| Autor: | Eva Kutejová, Gabriela Ondrovičová, Ľuboš Ambro, Jana Bellová, Jacob A. Bauer, Vladimír Pevala, Nina Kunová |
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| Rok vydání: | 2014 |
| Předmět: |
Conformational change
Protease La medicine.medical_treatment ATPase Molecular Sequence Data Mutant Biochemistry Adenosine Triphosphate ATP hydrolysis Catalytic Domain medicine Humans Amino Acid Sequence Molecular Biology Protease biology Mutagenesis Caseins Active site Cell Biology Enzyme Activation Mechanism of action Mutation biology.protein medicine.symptom Protein Binding |
| Zdroj: | FEBS Journal. 281:1784-1797 |
| ISSN: | 1742-464X |
| DOI: | 10.1111/febs.12740 |
| Popis: | 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 β-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 β-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 β-casein. Finally, we also noted that Lon's ability to digest small peptides can be inhibited by moderate ATP concentrations. Database Lon (Endopeptidase La), EC 4.4.21.53 Structured digital abstract • hLonP cleaves beta casein by protease assay (1, 2, 3, 4, 5, 6) • hLon and hLon bind by cross-linking study (View interaction) |
| Databáze: | OpenAIRE |
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