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

Teresa Mc Cormack, Wolfgang Baumeister, Louis Grenier, Carolyn Moomaw, Louis Plamondon, Bikash Pramanik, Clive A. Slaughter, François Soucy, Ross Stein, Frank Zühl, Lawrence Dick

Research output: Contribution to journalArticle

56 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)26103-26109
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number42
DOIs
StatePublished - Oct 17 1997
Externally publishedYes

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Rhodococcus
Lactones
Proteasome Endopeptidase Complex
Catalytic Domain
Kinetics
Peptides
Boronic Acids
Proteasome Inhibitors
Peptide Mapping
Protein Sequence Analysis
Threonine
Amino Acids
Aldehydes
Molecules
Hydroxyl Radical
High performance liquid chromatography
Titration
Stoichiometry
High Pressure Liquid Chromatography
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Mc Cormack, T., Baumeister, W., Grenier, L., Moomaw, C., Plamondon, L., Pramanik, B., ... Dick, L. (1997). Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. Journal of Biological Chemistry, 272(42), 26103-26109. https://doi.org/10.1074/jbc.272.42.26103

Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. / Mc Cormack, Teresa; Baumeister, Wolfgang; Grenier, Louis; Moomaw, Carolyn; Plamondon, Louis; Pramanik, Bikash; Slaughter, Clive A.; Soucy, François; Stein, Ross; Zühl, Frank; Dick, Lawrence.

In: Journal of Biological Chemistry, Vol. 272, No. 42, 17.10.1997, p. 26103-26109.

Research output: Contribution to journalArticle

Mc Cormack, T, Baumeister, W, Grenier, L, Moomaw, C, Plamondon, L, Pramanik, B, Slaughter, CA, Soucy, F, Stein, R, Zühl, F & Dick, L 1997, 'Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism', Journal of Biological Chemistry, vol. 272, no. 42, pp. 26103-26109. https://doi.org/10.1074/jbc.272.42.26103
Mc Cormack T, Baumeister W, Grenier L, Moomaw C, Plamondon L, Pramanik B et al. Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. Journal of Biological Chemistry. 1997 Oct 17;272(42):26103-26109. https://doi.org/10.1074/jbc.272.42.26103
Mc Cormack, Teresa ; Baumeister, Wolfgang ; Grenier, Louis ; Moomaw, Carolyn ; Plamondon, Louis ; Pramanik, Bikash ; Slaughter, Clive A. ; Soucy, François ; Stein, Ross ; Zühl, Frank ; Dick, Lawrence. / Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and mechanism. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 42. pp. 26103-26109.
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