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

S. G. Rudyak, Michael D. Brenowitz, T. E. Shrader

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)9317-9323
Number of pages7
JournalBiochemistry
Volume40
Issue number31
DOIs
StatePublished - Aug 7 2001

Fingerprint

Protease La
Mycobacterium smegmatis
Oligomerization
Magnesium Chloride
Catalyst activity
Peptide Hydrolases
Caseins
Protein Unfolding
Association reactions
Sedimentation
Proteins
Adenylyl Imidodiphosphate
Ions
Experiments
Ultracentrifugation
Enzymes
Edetic Acid
Tryptophan
Dimers
Trypsin

ASJC Scopus subject areas

  • Biochemistry

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Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. / Rudyak, S. G.; Brenowitz, Michael D.; Shrader, T. E.

In: Biochemistry, Vol. 40, No. 31, 07.08.2001, p. 9317-9323.

Research output: Contribution to journalArticle

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