Evidence for hemiketals as intermediates in the inactivation of serine proteinases with halomethyl ketones

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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., the inductive substituent constant of X, reveals a linear relationship between the free energy of binding and the electron-withdrawn power of X. The reversible binding constant obtained for the corresponding chloromethyl ketone Lys-Ala-LysCH2Cl falls on this line, indicating that the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.