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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.Lon (Endopeptidase La), EC 188.8.131.52• hLonPcleavesbeta casein by protease assay (1, 2, 3, 4, 5, 6)• hLon and hLonbind by cross-linking study (View interaction)The proteolytic active site of the ATP-dependent, human mitochondrial Lon protease contains two conserved glycine residues, Gly893 and Gly894, which appear both to be involved in the conformational changes that occur upon substrate binding and to influence ATPase activity. The loop containing these residues must move during digestion of larger protein substrates, but not during cleavage of smaller peptides.