Mg²⁺-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg²⁺ ions and stimulated by unfolded proteins such as α-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg²⁺. Addition of α-casein partially substitutes for Mg²⁺ in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of α-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg²⁺ ions but not by AMP-PNP or ATPγS. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl₂. Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl₂, at pH 8.0 and 20 °C, and that the assembly reaction is Mg²⁺ dependent; the mole fraction of hexamer decreases with decreasing MgCl₂ to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl₂ concentrations yields two assembly models: dimer ↔ tetramer ↔ hexamer and timer ↔ hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg²⁺-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg²⁺, and stimulator, unfolded protein.