Peptidyl boronates inhibit Salmonella enterica serovar Typhimurium Lon protease by a competitive ATP-dependent mechanism

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.