Histidine ionizing groups

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

With multiple ionizable groups, such as in amino acids and proteins, each group titrates separately according to its pKa. The titration curves shown in Fig. 23-5 are for the amino acids glycine, histidine, and glutamate. 

Some amino acids have additional ionizable groups in their side-chains. These may be acidic or potentially acidic (aspartic acid, glutamic acid, tyrosine, cysteine), or basic (lysine, arginine, histidine). We use the term potentially acidic to describe the phenol and thiol groups of tyrosine and cysteine respectively under physiological conditions, these groups are unlikely to be ionized. It is relatively easy to calculate the amount of ionization at a particular pH, and to justify that latter statement. 

These proteins may also have an ionizable group with a pKa of 8.0, perhaps from one of the histidines that is linked to the oxidation-reduction reaction. 

Despite the fact that carbonic anhydrase was the first zinc metalloenzyme identified1233 and a good deal is known of its structure, there is still controversy about the nature of the various active-site species and the detailed mechanisms of their action. In particular, the identity of the group with a pXa of 7 that is involved in the mechanism, and the stereochemistry around the zinc ion during catalysis, are still in dispute. The various mechanisms proposed assume either ionization of a histidine imidazole group (bound or not to the zinc) and nucleophilic attack on C02 by the coordinated imidazolate anion,1273,1274 or ionization of the Znn-coordinated water and nucleophilic attack on C02 by OH. 1271 Many papers on this problem have appeared recently and the extensive literature is the subject of the several review articles referred to above. 

Titration curve of /3-lactoglobulin. At very low values of pH (<2) all ionizable groups are protonated. At a pH of about 7.2 (indicated by horizontal bar) 51 groups (mostly the glutamic and aspartic amino acids and some of the histidines) have lost their protons. At pH 12 most of the remaining ionizable groups (mostly lysine and arginine amino acids and some histidines) have lost their protons as well. 

The activities of Fe— and Mn—SODs are decreased above pH 8.5, consistent with the involvement of an ionizable group of the protein with a pKa value between 9 and 10 in the catalytic cycle.77 The most likely residue appears to be Tyr-34, which is only 5 A apart from the Mn.51 A possible route of O2 to the Mn site in T. thermophilus Mn-SOD50 runs across the helix 1 between the Lys-29 and Tyr-34, and the O2 binds to the Mn3+ tram to Asp-163 with octahedral coordination (Figs. 10.7 and 10.8). The route to the metal is lined by aromatic and histidine residues including His-26, His-30, Tyr-34, His-81, Phe-84, Trp-165 and His-167. 

Ionization State of Histidine Each ionizable group of an amino acid can exist in one of two states, charged or neutral. The electric charge on the functional group is determined by the relationship between its pfCa and the pH of the solution. This relationship is described by the Henderson-Hasselbalch equation. 

The active site has a zinc(II) ion bonded to three histidine imidazole groups and a fourth site occupied by water or hydroxide ion, L3Zn OH, where L = imidazole N from histidine. Experimentally, the enzyme loses activity below pH 7, indicating that an ionizable group of pK 1 is part of the active site. In addition, it is known that the product of the hydration of CO2 is HCO3 , would be expected in neutral or basic solution. 

Hemoglobin contains many ionizable groups. One in particular is a histidine residue that has a pK around neutrality. Thus, at any time, hemoglobin is also present as a mixture of protonated and unprotonated forms. The proportion of each depends on the pH of the blood. 

Two kinetic methods have been used to identify catalytic groups from pH profiles. First, the temperature coefficients of the pK values of the various ionizing groups differ considerably. Carboxyl and phosphate groups have A//jo values near zero, whereas histidine, cysteine, and tyrosine groups have values of... 

The interactions of the amino acid derivatives with a modified C18 column and micellar mobile phases of the anionic SDS are mainly of hydrophobic and electrostatic nature. The basic structure of the amino acid isoindoles is the same. Consequently, in the absence of electrostatic interactions, the hydrophobic character of the Rj substituent should be responsible of the retention. The nitrogen atom in the isoindole heterocycle is nonprotonated at pH 3, and thus, electrostatic interactions can only exist with ionizable groups giving a positive charge to R in the molecule. This occurs with arginine and histidine, which have amino groups that do not react with OPA. 

---