title: Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities.
abstract: 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.
  
  
  METHODS
  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).
authors: Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
authors: Ľuboš
authors: Ľ
authors: Ambro
authors: Vladimír
authors: V
authors: Pevala
authors: Gabriela
authors: G
authors: Ondrovičová
authors: Jana
authors: J
authors: Bellová
authors: Nina
authors: N
authors: Kunová
authors: Eva
authors: E
authors: Kutejová
authors: Jacob
authors: J
authors: Bauer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D049070:Protease La / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D002364:Caseins; D000255:Adenosine Triphosphate; D049070:Protease La
keywords: ATP-dependent protease; glycine loop; human Lon protease; mutagenesis; peptidase