States of amino acid residues in proteins

Abstract

Abstract The ionizations of tyrosine residues of chymotrypsinogen, α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4) were measured spectrophotometrically as a function of pH, immediately after, and several hours after, addition of alkali to protein solutions, and the ionization characteristics of various states of their tyrosine residues were estimated from the results. The four residues of both chymotrypsinogen and α-chymotrypsin were classified into two rapidly ionizing, one slowly ionizing and one non-ionizable residues. However, considerable differences were found in the degree of binding or the pK value between their residues. Ten tyrosine residues in the trypsin moleculr were grouped into four slowly ionizing residues with pK = 10.8 and m (the number of hydroxyl ions involved in the ionization) = 2.0, and six rapidly ionizing residues with pK = 10.0 and m = 1.0. The pH-activity curve for trypsin indicated the deactivation by alkali with the same values of pK and m as for the ionization of the four bound residues. The effect of temperature on the activity curve, and the time course of the activity drop during the incubation with alkali, indicated a close correlation between the ionization of the bound tyrosine residues and the deactivation. The correlation similarly examined for α-chymotrypsin was less satisfactory. A model for the active center of trypsin is proposed to account for both the close correlation found in the present study and the concerted action of serine and histidine residues previously established.