NUCLEAR MAGNETIC RESONANCE STUDIES OF THE ACTIVE SITE OF BOVINE CHYMOTRYPSINOGEN A AND CHYMOTRYPSIN A(ALPHA)

IGNACIO IBANEZ, Purdue University

Abstract

Reversible unfolding of bovine chymotrypsinogen A in ('2)H(,2)O either by heating at low pH or by exposure to 6M guanidinium chloride results in exchange of virtually all the nitrogen bound hydrogens that give rise to low-field ('1)H NMR peaks. These pre-exchange procedures have enabled the resolution of two peaks attributed to the two histidine residues of chymotrypsinogen A. Assignments of the C('(epsilon)) hydrogen peaks to histidine-40 and histidine-57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidine C('(epsilon))-H peaks were also resolved with chymotrypsin A(,(alpha)) prepared by activation of pre-exchanged zymogen or by direct pre-exchange of chymotrypsin A(,(alpha)) followed by short incubation at high pH. The histidine peaks of chymotrypsin A(,(alpha)) were assigned by comparison to chymotrypsin A(,(alpha)) reacted with N('(alpha))-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Alkylation of histidine-57 by TPCK results in the disappearance of one peak, which is assigned to this residue, without affecting the other histidine peak, which is assigned to histidine-40. The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with the neighboring carboxyl, aspartate-102 in case of histidine-57 and aspartate-194 in the case of histidine-40. In bovine chymotrypsinogen A in ('2)H(,2)O at 31(DEGREES)C, histidine-57 has a pK(,a)('') of 7.3, and the transition involving aspartate-102 has a pH(,mid) of 1.4; 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 titration curve of histidine-57 has two transitions with midpoints at pH 6.5 and 2.9, and histidine-40 has a pK(,a)('') of 7.3. The results suggest that the pK(,a)('') of histidine-57 is higher than the pK(,a)('') of aspartate-102 in both the zymogen and enzyme. The ('1)H NMR chemical shift of the C('(epsilon))-H of histidine-57 in the chymotrypsin A(,(alpha)):bovine pancreatic trypsin inhibitor complex is constant betweeen pH 4 and 9 at a value similar to that of the protonated form of histidine-57 in the free enzyme, suggesting that a proton is added to histidine-57 upon complex formation. ('31)P NMR spectra of the diisopropyl phosphoryl (iPr(,2)P) derivative of chymotrypsinogen A reveal that the phosphorus chemical shift is perturbed by changes in pH. The ('31)P NMR titration curve shows two transitions with midpoints at pH 3.3 and 7.6. These values correlate well with the values obtained for the C('(epsilon))-H peak of histidine-57 in ('1)H NMR spectra of the same derivative. The ('31)P NMR titration curve of iPr(,2)P-chymotrypsin A(,(delta)) also reveals two transitions with inflections at pH 7.5 and 4.3. 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(,a)('') values, the chemical shift of histidine-57, which is deshielded by 0.6 ppm in the zymogen, becomes normalized upon activation. Also the ('31)P NMR chemical shift of the iPr(,2)P-enzyme is deshielded as compared to the iPr(,2)P-zymogen. These changes may explain part of the increase in catalytic activity upon activation.

Degree

Ph.D.

Subject Area

Biochemistry

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