Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones

Biochemistry. 1994 Apr 5;33(13):3934-40. doi: 10.1021/bi00179a020.

Abstract

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease in monocytes that is essential for the proteolytic activation of interleukin-1 beta, an important mediator of inflammation. Peptide (acyloxy)methyl ketones designed with the appropriate peptide recognition sequence (Ac-Tyr-Val-Ala-Asp-CH2-OC(O)Ar) are potent, competitive, irreversible inhibitors. Mass spectrometry and sequence analysis indicate that inactivation proceeds through expulsion of the carboxylate leaving group to form a thiomethyl ketone with the active site Cys285. The second-order inactivation rate is independent of leaving group pKa, with an approximate value of 1 x 10(6) M-1 s-1. This rate constant is directly proportional to the reaction macroviscosity, indicating that the rate-limiting step in inactivation is association of enzyme and inhibitor, rather than any bond-forming reactions. Affinity labeling of THP.1 monocytic cell cytosol with a biotinylated tetrapeptide (acyloxy)methyl ketone for 28 half-lives resulted in labeling of only ICE, demonstrating the selectivity of these inhibitors. These inhibitors are relatively inert toward other bionucleophiles such as glutathione (< 5 x 10(-4) M-1 s-1), making them excellent candidates for in vivo studies of enzyme inhibition.

MeSH terms

  • Amino Acid Sequence
  • Caspase 1
  • Cysteine Proteinase Inhibitors / chemistry*
  • Humans
  • Ketones / chemistry
  • Kinetics
  • Metalloendopeptidases / antagonists & inhibitors*
  • Molecular Sequence Data
  • Monocytes / enzymology
  • Oligopeptides / chemistry
  • Structure-Activity Relationship

Substances

  • Cysteine Proteinase Inhibitors
  • Ketones
  • Oligopeptides
  • Caspase 1
  • Metalloendopeptidases