Mechanism of hydrolysis by serine proteases: direct determination of the pKa's of aspartyl-102 and aspartyl-194 in bovine trypsin using difference infrared spectroscopy.

The pKa of aspartyl-102 in trypsin is shown to be 6.8+/-0.2 by difference infrared titration. All but 2.5 of the carboxyls in bovine trypsin were first modified with semicarbazide. The modified enzyme still retains full activity toward nonspecific substrates. The remaining free carboxyls include one equivalent each of Asp-102 and Asp-194. The absorbances associated with the C=O and C=O stretching modes at 1570 and 1710 cm-1 were used to monitor the proportion of ionized or protonated carboxyl present in the enzyme as a function of pD. The pKa of 6.8 was assigned to Asp-102 using copper ions that bind to trypsin between Asp-102 and histidine-57, so lowering the pKapp of Asp-102. The implication of this result for the ionization of the active site, and for the mechanism of serine proteases, is discussed. Asp-194 and the C terminus are shown to titrate with an average pKa of 2.9.