Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin A.alpha.

Abstract

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the N-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring C.epsilon.1 hydrogens. These preexchange procedures have enabled the resolution of 2 peaks, using 250 MHz correlation 1H NMR spectroscopy, that are attributed to the 2 histidyl residues of chymotrypsinogen A. Assignments of the C.epsilon.1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl C.epsilon.1 H peaks were also resolved with solutions of preexchanged chymotrypsin A.alpha.. The histidyl peaks of chymotrypsin A.alpha. were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit 2 inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 .degree.C, histidine-57 has a pK'' of 7.3 and aspartate-102 a pK'' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin A.alpha. under the same conditions, the histidine-57-aspartate-102 system has pK'' values of 6.1 and 2.8, and histidine-40 has a pK'' of 7.2. Apparently the pK'' of histidine-57 is higher than the pK'' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK'' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the C.epsilon.1 H of histidine-57 in the chymotrypsin A.alpha.-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex, suggesting that the mechanisms of interaction are similar in the 2 complexes.