Mg2+-linked oligomerization modulates the catalytic activiy 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 a-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is depen dent upon both its concentration and that of Mg2+. Addition of a-casein par tially substitutes for Mg2+ in activating the enzyme. In chemical dissociat ion experiments, higher concentrations of urea were required to inhibit Ms- Lon's catalytic activities after an addition of a-casein. Analytical ultrac entrifugation was used to directly probe the effect of activators of peptid ase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeri c forms of the protein and that the self-association reaction is facilitate d by Mg2+ ions but not by AMP-PNP or ATP gammaS. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrat ions of MgCl2. Sedimentation equilibrium analysis shows that Ms-Lon associa tes to a hexamer at 50 mM Tris and 10 mM MgCl2 at pH 8.0 and 20 degreesC, a nd that the assembly reaction is Mg2+ dependent; the mole fraction of hexam er decreases with decreasing MgCl2 to undetectable levels in 10 mM EDTA. Th e analysis of experiments conducted at a series of initial protein and MgCl 2 concentrations yields two assembly models: dimer H tetramer H hexamer and timer H hexamer, equally consistent with the data. Limited trypsin digesti on, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg2+-linked oligomerization. These results sho w 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.