PINA INHIBITS ATP HYDROLYSIS AND ENERGY-DEPENDENT PROTEIN-DEGRADATIONBY LON PROTEASE

The bacteriophage T4 PinA protein inhibited degradation of [H-3]alpha- methyl casein by purified Lon protease from Escherichia coli, but inhi bition was noncompetitive with respect to casein, PinA did not inhibit cleavage of the fluorogenic peptide, yl-alanylalanylphenylalanyl-3-me thoxynaphthylamide and, moreover, did not block the ability of protein substrates, such as casein, to activate cleavage of fluorogenic pepti des by Lon, Thus, PinA does not block the proteolytic active site or t he allosteric protein-binding site on Lon, Inhibition of basal ATPase activity was variable (50-90%), whereas inhibition of protein-activate d ATPase activity was usually 80-95%, Inhibition was noncompetitive wi th respect to ATP, PinA did not block activation of peptide cleavage b y nonhydrolyzable analogs of ATP, These data suggest that PinA does no t bind at the ATPase active site of Lon and does not interfere with nu cleotide binding to the enzyme, PinA inhibited cleavage of the 72-amin o acid protein, CcdA, degradation of which requires ATP hydrolysis, bu t did not inhibit cleavage of the carboxyl-terminal 41-amino acid frag ment of CcdA, degradation of which does not require ATP hydrolysis, Pi nA thus appears to interact at a novel regulatory or enzymatic site in volved in the coupling between ATP hydrolysis and proteolysis, possibl y blocking the protein unfolding or remodeling step essential for degr adation of high molecular weight protein substrates by Lon.