title: Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones.
abstract: The mechanism of inactivation of serine proteinases by peptide halomethyl ketone inhibitors was studied through the inhibition of trypsin with a series of model peptide ketones (Lys-Ala-LysCH2X). In this series, X is a poor leaving group with increasing electron-withdrawing capacity (X = H, CH2CO2CH3, COCH3, OCOCH3, and F), and as expected, the peptide ketones are reversible, competitive inhibitors of trypsin. The strength of binding of these inhibitors to trypsin increases with the electron-withdrawing ability of X, indicating that the inhibition constant Ki obtained is a measure of reversible hemiketal formation between the inhibitor ketone carbonyl group and the hydroxyl group of the active site serine. A Hammett plot of -log Ki vs. sigma I, the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawing power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the reversible binding involves hemiketal formation, which is followed by alkylation of the enzyme.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000138:chemical synthesis* / Q000494:pharmacology; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D010450:Endopeptidases / Q000378:metabolism*; D005463:Fluoroacetates / Q000138:chemical synthesis* / Q000494:pharmacology; D007202:Indicators and Reagents; D007659:Ketones; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000494:pharmacology; D011485:Protein Binding; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000138:chemical synthesis*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D005463:Fluoroacetates; D007202:Indicators and Reagents; D007659:Ketones; D009842:Oligopeptides; D014361:Trypsin Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin