Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism

We have studied the mechanism of inhibition of the recombinant Rhodococcus proteasome by four different chemical classes of active site- directed small molecule inhibitors. Clasto-lactacystin β-lactone is a time- dependent inhibitor of the Rhodococcus proteasome's ability to hydrolyze Suc- Leu-Leu-Val-Tyr-AMC, a substrate for this proteasome's single type of active site, and proceeds with a k(inact)/[I] of 1,700 M^-1 s^-1. Using peptide mapping of tryptic digests, LC/MS, and amino acid sequence analysis, we have established that the Oγ of the hydroxyl group on the N-terminal threonine of the β-subunit is the sole modification made by the β-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per β-subunit. Prior modification with β-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the Oγ moiety on Thr¹. High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome β-subunit and its tryptic peptides suggests that the peptidyl vinyl sulfone modifies a residue in the N-terminal 20 amino acids. This modification is also blocked by prior treatment with β-lactone.