TY - JOUR
T1 - Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism
AU - Mc Cormack, Teresa
AU - Baumeister, Wolfgang
AU - Grenier, Louis
AU - Moomaw, Carolyn
AU - Plamondon, Louis
AU - Pramanik, Bikash
AU - Slaughter, Clive A.
AU - Soucy, François
AU - Stein, Ross
AU - Zühl, Frank
AU - Dick, Lawrence
PY - 1997/10/17
Y1 - 1997/10/17
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.272.42.26103
DO - 10.1074/jbc.272.42.26103
M3 - Article
C2 - 9334174
AN - SCOPUS:0030671540
SN - 0021-9258
VL - 272
SP - 26103
EP - 26109
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 42
ER -