Carbon nuclear magnetic resonance studies of the histidine residue in α-lytic protease. Implications for the catalytic mechanism of serine proteases

Abstract

Selective ^(13)C enrichment of C-2 of the single histidine
\nresidue of the serine protease α-lytic protease has allowed direct study of the Asp-His-Ser catalytic triad. Both the chemical shift of C-2 and the coupling between C-2 and its directly bonded hydrogen have been observed as a function of pH. We interpret the results to indicate that only below
\npH 4 does the imidazole ring of the histidine residue become
\nprotonated and only above pH 6.7 does the aspartic acid residue lose a proton to generate a carboxylate anion. Thus,
\nover the pH range 4-6.7, the catalytic triad consists of a neutral aspartic acid and a neutral histidine residue-not the ionized forms hitherto assumed. These new assignments for the ionization characteristics of the aspartic acid and histidine residues of the catalytic triad lead to a proposed catalytic mechanism that avoids any requirement for unfavorable charge separation. In this view, the histidine residue plays two roles: (i) it provides insulation between water and the buried carboxylate anion of the aspartate, thus ensuring the carboxylate group a hydrophobic environment, and (ii) it provides a relay for net transfer of protons from the serine hydroxyl to the carboxylate anion. The aspartate anion acts as the ultimate base which holds a proton during catalysis. An anionic, rather than a neutral, base has advantages; it both avoids the necessity of charge separation and, by giving the catalytic locus an overall negative charge, assists preferential expulsion of product relative to substrate from the active site of the enzyme. Relaxation measurements (T_I, T_2, and nuclear Overhauser enhancement) indicate that, over the pH range of enzymic activity, the histidine residue is held rigidly within the protein.