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 Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) 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 alpha-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 Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) 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 Mg(2+)-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, Mg(2+), and stimulator, unfolded protein.
Rudyak
S G
SG
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Brenowitz
M
M
Shrader
T E
TE
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D015636:Magnesium Chloride / Q000378:metabolism; D008956:Models, Chemical; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation
D002364:Caseins; D020536:Enzyme Activators; D006360:Heat-Shock Proteins; D015636:Magnesium Chloride; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D000251:Adenosine Triphosphatases; D008274:Magnesium