Polypeptide stimulators of the Ms-Lon protease

Both the peptidase activity against small fluorescent peptide substrates an d the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as alpha-casein. This stimulation reveals the simultaneous in teraction of Lon with two proteolytic substrates-alpha-casein and the pepti de substrate. To understand the cellular function of this stimulation, it i s important to determine the physical properties of Lon stimulators. The ab ilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from M ycobacterium smegmatis (Ms-Lon) and its N-terminal truncation mutant (N-E22 6) were determined. We report that cationic but not anionic rcAAs stimulate d Rls-Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the de gree of hydrophobicity of these polypeptides and reached levels >10-fold hi gher than observed previously for Ms-Lon stimulators such as alpha-casein. In contrast to alpha-casein, which stimulates Ms-Lon's peptidase activity b y 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity with out concomitant stimulation of ATPase activity. Active site labeling experi ments suggested that both rcAAs and ATP increased peptidase activity by inc reasing accessibility to the peptidase active site. Peptidase activity assa ys in the presence of both alpha-casein and rcAAs revealed that interaction s of rcAAs and alpha-casein with Ms-Lon are extremely complex and not mutua lly exclusive. Specifically, (1) additions of low concentrations of alpha-c asein (<50 mu g/mL) caused a further stimulation of Ms-Lon's rcAA-stimulate d peptidase activity; (2) additions of higher concentrations of alpha-casei n inhibited Ms-Lon's rcAA-stimulated peptidase activity; (3) additions of a ll concentrations of alpha-casein inhibited N-E226's rcAA-stimulated peptid ase activity. We conclude the Ms-Lon can interact with an rcAA, alpha-casei n, and a substrate peptide simultaneously, and that formation of this quate rnary complex requires the N-terminal domain of Ms-Lon. These data support models of Ms-Lon that include two allosteric polypeptide binding sites dist inct from the catalytic peptidase site.