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 beta-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 O gamma of the hydroxyl group on the N terminal threonine of the beta-subunit is the sole modification made by the beta-lactone. Active site titrations of the Rhodococcus proteasome with reversible peptide aldehydes show the expected stoichiometry of one inhibitor molecule per beta-subunit. Prior modification with beta-lactone completely abrogates the binding of peptidyl boronic acid inhibitors, suggesting that these inhibitors also inactivate the enzyme by reacting with the O gamma moiety on Thr(1), High performance liquid chromatography analysis of peptidyl vinyl sulfone-modified intact Rhodococcus proteasome beta-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 beta-lactone.