Ionization Behavior of the Histidine Residue in the Catalytic Triad of Serine Proteases: MECHANISTIC IMPLICATIONS

Michael W Hunkapiller,Stephen H Smallcombe,Don R Whitaker,John H Richards

doi:10.1016/s0021-9258(19)43228-6

Michael W Hunkapiller, Stephen H Smallcombe + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(19)43228-6

Copy DOI

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.

Highlights

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
‘H-o during catalysis, we sought to determine the state of ionization of the imidazole ring as a function of pH
The protein investigated was oc-lytic protease which we chose for a variety of reasons. (a) a-Lytic protease, in binding and catalysis, displays the characteristics of other serine proteases- of elastase-and can, serve as a paradigm of these enzymes [6,7,8,9,10,11]. (b) It possesses only a single histidine residue which belongs to the catalytic triad and whose properties can be unambiguously studied

Summary

SUMMARY

A-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. Magnetic resonance studies of the chemical shift and coupling constant (IJoH) behavior of this nucleus as a function of pH suggest that the imidazole ring is neutral above pH 5 and that the group which is known to ionize with pK, 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.

Institutes of Health Traineeship

PROCEDURE

Findings

Cation Neutral