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

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

Mechanism of serine protease action. Ionization behavior of tetrahedral adduct between α-lytic protease and tripeptide aldehyde studied by carbon-13 magnetic resonance

Magnetic resonance techniques have been used to study the ionization behavior of the catalytic triad of the serine protease, α-lytic protease, in the tetrahedral, hemiacetal complex it forms with the aldehyde inhibitor, N-ac-L-ala-L-pro-L-alaninal. Chemical shift, coupling constant and relaxation measurements of a carbon-13 nucleus specifically incorporated in C-2 of the imidazole ring of the single histidine residue of the protein show that, above pH 7, the imidazole ring of the catalytic triad in the enzyme + aldehyde complex is neutral. We suggest, further, that a neutral carboxylic acid group for Asp 102 and an oxyanion for the hemiacetal are most likely to describe the state of ionization of the other groups above pH 7. Around pH 6·25, both the oxyanion and the histidine become protonated in a co-operative process which forces the histidine away from its rigidly localized position as a member of the catalytic triad into a solution-like environment

Magnetic resonance studies of protein-small molecule interactions. Binding of N-trifluoroacetyl-D-(and L-)-tryptophan to α-chymotrypsin

A magnetic resonance technique has been developed for studying the competitive binding to proteins of two small molecules; the nmr spectrum of only one needs to be observed. This technique has been applied to study the competition between N-trifluoroacetyl-D-tryptophan and the L enantiomer for the active site of α-chymotrypsin from pH 5.0 to 8.0. The chemical shift for the fluorine nuclei of N-trifluoroacetyl-D-tryptophan bound to the enzyme is found to be the same as that for N-trifluoroacetyla-D -p fluorophenylalanine. The binding of both D- and L-tryptophan derivatives shows a marked dependence on deprotonation of a group on the free enzyme with pK_a = 6.6 (presumably His-57)

Magnetic resonance studies of protein-small molecule interactions. Binding of N-trifluoroacetyl-D-(and L-)-p-fluorophenylalanine to α-chymotrypsin

Magnetic resonance studies of the interaction of N-trifluoroacetyl-n-(and L-)p-fluorophenylalanine with α-chymotrypsin have been used to investigate the molecular details of the enzyme-inhibitor interaction including the effect of pH (from 5.0 to 8.0). The principles of the technique are described. We conclude that the trifluoroacetyl group of then isomer interacts with the catalytic locus (His-57, Ser-195) while that of the L isomer is directed toward Ser-214. The aromatic ring of both then and L isomer resides in the hydrophobic pocket. The binding constant for the n isomer increases with neutralization of a group which has pK. of 6.6 in the free-enzyme (presumably His-57). The dimerization of chymotrypsin strongly affects the quantitative results and has been explicitly included in the analysis

Binding Dynamics in Biological Systems. Interaction of MOPC-315 with ^(19)F Labelled Nitrophenyl Haptens

We have studied the dynamics of binding of ^(19)F labelled haptens by mouse plasmacytoma antibody MOPC-315, a protein which shows specificity for nitrophenyl haptens. The off-rates for the dissociation of hapten from MOPC-315 proteins (7S, Fab′ and Fv) were determined by the application of several methods including a technique which, in certain cases, allows the direct determination of the rate of exchange of nuclei between magnetically non-equivalent sites without requiring prior knowledge of intrinsic line widths. Used in conjunction with independently determined data on line widths, chemical shifts, and binding affinities, these studies show that the rates for hapten association to, or dissociation from, the intact 7S antibody, the Fab′ fragment, and the Fv fragment of MOPC-315 are essentially the same. They also indicate the presence, probably in the Fv region, of a low affinity (K∼ 10^3M^(−1)) site(s) for hapten binding. The mobility of the hapten combining site decreases as the size of the protein increases. These rate data, which were determined at relatively high protein concentrations (up to 40 mg ml^(−1)), agree with on-rates determined at much lower protein concentrations (≤1 mg ml^(−1)); we therefore conclude that protein aggregation, if it does occur, does not significantly affect binding in these systems