The Reactivity of the N-terminal Isoleucine of α-Chymotrypsin as a Function of Ionic Strength

Abstract

The reactivity of the α-amino group of isoleucine-16 of α-chymotrypsin towards nitrous acid at pH 4.0 and 0° is strongly dependent on ionic strength. Third-order deamination rate constants at low ionic strength (μ = 0.1 M) are 500–1000 times higher than those at high ionic strength (μ = 5.0 and 6.0 M) and are independent of the nature of the ions and the chymotrypsin concentration. Extrapolation to zero ionic strength of a plot of the logarithms of the constants against ionic strength leads to a value which is the same as that for the exposed α-amino group of the model compounds isoleucylvaline and valylvaline, and of the N-terminal isoleucine of pepsin. The deamination rate constant of the dipeptide valylvaline varies only two- to three-fold between ionic strength of 0.1 M and 6 M. The results suggest that the concept of a "buried" N-terminal as shown by X-ray analysis (carried out at pH 4.2 and ionic strength 9–11 M) requires modification; at low ionic strength (0.1 M) the reactivity of the N-terminal is only little below that of an exposed amino group, a fact which suggests that the amino group is much more available than shown by the X-ray analysis. The results are interpreted in terms of an effect of the ionic strength on the equilibrium between two conformational states of the enzyme.

Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

“…The reactivity of isoleucine in trypsin under conditions comparable to those of a-chymotrypsin is about 10 times higher and that of thrombin about 50 times higher. Possible interpretations of such differences have been discussed extensively in a previous publication (9) in connection with the effect of the ionic strength on the reactivity of the isoleucine in a-chymotrypsin. It was pointed out there that the differences in reaction rates can be interpreted in three ways: (a) a "loosening" of the structure around the reactive residue allowing limited access to the reagent, (b) a rupture of the ion pair interaction with a change in the conformation which brings the N-terminal out into solution, or (c) a partial unfolding of the molecule which leaves the ion pair essentially intact but allows unrestricted access of the reagent to the amino group.…”

“…These were carried out as described previously (6,9), except that in all cases H2S04 was used for adjusting the pH. At the end of the reaction, the proteins and poly-amino acids were precipitated with 20 volumes of a mixture of ethanol -acetone-diethyl ether (10: 10 : 1 by volume) at -15".…”

“…An amino acid analysis showed that even after reaction periods of 2 days at room temperature all the tyrosine could be The Reaction of Nitrous Acid with Ami~zo Acid Residues in Proleins The purpose of the experiments described in this paper was to characterize qualitatively and quantitatively the reaction of nitrous acid with amino acid residues and amino acid derivatives in order to evaluate it as a probe for various residues in proteins. Earlier publications (6, 9) have shown that the reagent can yield very interesting information when used as a probe for N-terminal residues. Table 4 are the same as those of dipeptides (after cos-trypsinsgen, the reaction of only one tryptophan rection for ionic strength effects) shows that out of four has so far been detected, The reaction the protein has no effect on the reactivity of a rate constant is about 1/100 of that of a model fully exposed group; on the sther hand, the compound.…”

“…During the study of the action of nitrous acid on serine proteinases, very large differences were observed in the reactivities of the N-terminal groups. Thus for instance, the reactivity of the amino group of isoleucine-16 in a-chymotrypsin is profoundly aiTectd by ionic strength (9). Under otherwise identical conditions, the reactivities of homologous N-terminal groups of trypsin, a-chymotrypsin, elastase, and thrombin show differences of over two orders of magnitude.…”