Ionization behavior of the histidine residue in the catalytic triad of serine proteases

Abstract

α-Lytic protease is a homologue of the mammalian serine proteases such as trypsin, chymotrypsin, and elastase, and its single histidine residue belongs to the Asp-His-Ser catalytic triad. This single histidine residue has been selectively enriched in the C-2 carbon with 13C. Magnetic resonance studies of the chemical shift and coupling constant (1Jch) behavior of this nucleus as a function of pH suggest that the imidazole ring is neutral above pH 5 and therefore that the group which is known to ionize with pKa near 6.7 must be the aspartic acid residue. Implications of these new pKa assignments for the catalytic mechanism of serine proteases are discussed and include the absence of any need to separate charge during catalysis. The histidine residue plays two roles. (a) It insulates the aspartic acid from an aqueous environment and accordingly raises its pKa. (b) It serves as a bidentate base to accept a proton from the serine at one of its nitrogens and concertedly transfer a proton from its other nitrogen to the buried carboxylate anion during formation of the tetrahedral intermediate.