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

Mc Cormack, T., Baumeister, W., Grenier, L., Moomaw, C., Plamondon, L., Pramanik, B., et al. (1997). Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. Journal of Biological Chemistry., 272(42), 26103-26109.

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Mc Cormack, T., Author
Baumeister, W.1, Author              
Grenier, L., Author
Moomaw, C., Author
Plamondon, L., Author
Pramanik, B., Author
Slaughter, C., Author
Soucy, F., Author
Stein, R., Author
Zühl, F.1, Author              
Dick, L., Author
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1External Organizations, ou_persistent22              

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Free keywords: Binding Sites; *Cysteine Endopeptidases/de [Drug Effects]; *Cysteine Proteinase Inhibitors/pd [Pharmacology]; Kinetics; *Lactones/pd [Pharmacology]; *Multienzyme Complexes/de [Drug Effects]; Recombinant Proteins/de [Drug Effects]; *Rhodococcus/en [Enzymology]
 Abstract: 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 kinact/[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 Ogamma 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 Ogamma moiety on Thr1. 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.

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 Dates: 1997
 Publication Status: Published in print
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 Identifiers: eDoc: 318448
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Title: Journal of Biological Chemistry.
Source Genre: Journal
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Pages: - Volume / Issue: 272 (42) Sequence Number: - Start / End Page: 26103 - 26109 Identifier: -