Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones.
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.
Thornberry
N A
NA
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Peterson
E P
EP
Zhao
J J
JJ
Howard
A D
AD
Griffin
P R
PR
Chapman
K T
KT
D000595:Amino Acid Sequence; D020170:Caspase 1; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D006801:Humans; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000201:enzymology; D009842:Oligopeptides / Q000737:chemistry; D013329:Structure-Activity Relationship
D015853:Cysteine Proteinase Inhibitors; D007659:Ketones; D009842:Oligopeptides; D020170:Caspase 1; D008666:Metalloendopeptidases