Histidine ionization

Figure 3.15. Histidine Ionization. Histidine can bind or release protons near physiological pH.

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 ... 

Most frequently, protonation or deprotonation of protein occurs at ionizable, also called titratable, side chains, such as aspartate, glutamate or histidine. The ionization equilibrium of a titratable site,... 

The most significant amino acids for modification and conjugation purposes are the ones containing ionizable side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, and tyrosine (Figure 1.6). In their unprotonated state, each of these side chains can be potent nucleophiles to engage in addition reactions (see the discussion on nucleophilicity below). 

There are some side reactions that may occur when using EDC with proteins. In addition to reacting with carboxylates, EDC itself can form a stable complex with exposed sulfhydryl groups (Carraway and Triplett, 1970). Tyrosine residues can react with EDC, most likely through the phe-nolate ionized form of its side chain (Carraway and Koshland, 1968). The imidazolyl group of histidine may react with sulfo-NHS esters, resulting in an active carbonyl imidazole group which subsequently hydrolyzes (Cuatrecasas and Parikh, 1972). Finally, EDC may promote unwanted polymerization due to the usual abundance of both amines and carboxylates on protein molecules. 

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. 

The mechanism schematized above is a summary of the current knowledge. The role of Asp102 has long been controversial [10], Indeed, the catalytic triad has been depicted as a charge-relay system, meaning that the activation of the serine residue involves a concerted transfer of two protons, i.e., from serine to histidine and then to aspartic acid. More recent studies have shown that aspartic acid remains ionized and serves to stabilize the ionic transition state [6] [14-16],... 

In the late 1950s it was shown that imidazole catalyzes the hydrolyses of />-nitrophenyl acetate (7, 76) and that histidine was at the active site of a-chymotrypsin (2). These findings led Katchalski ei al. (39) to synthesize a number of histidine-containing polymers for evaluation as catalysts. Second-order rate constants were calculated on the basis of the concentration of neutral imidazole, that is, k2 = (A bs — .)/a[IM], where k , is the rate constant in the absence of catalyst and a is the fraction ionized. Some of these rate constants appear in Table I. All of the polymers possess less than... 

The crystal structure (Strop et al. 2001) reveals a homodimer with the zinc atom ligated by the sulfur atoms of two cysteines (Cys 32 and Cys 90) and the nitrogen atom of a histidine (His 87), as is the case for the plant-type enzyme (Fig. 11.3). The active site contains an HEPES buffer molecule in a position that implicates involvement of Asp 34 in the transport of protons after ionization of the zinc-bound water. 

Buffers resist changes in pH. Substances can act as buffers at their pK values. In proteins, the amino acids with ionizable R-groups can act as buffers, altiiough the only amino acid that is useful in maintaining physiologic pH (7.2-7.4) is histidine with an R-group pK near 7. Hemoglobin can act as an intracellular buffer in red blood cells because it contains histidyi residues. 

The structure of the ribosomal protein L9 from B. stearothermophilus is shown in Fig. 3. The folding kinetics and thermodynamics of its C-terminal domain have been studied as a function of pH by NMR and CD spectroscopies. The ionization state of the two histidines (Hisl06 and His 134) was found to be essential for the global stability and the folding rate of the protein. ... 

Studies on the effect of pH on peroxidase catalysis, or the heme-linked ionization, have provided much information on peroxidase catalysis and the active site structure. Heme-linked ionization has been observed in kinetic, electrochemical, absorption spectroscopic, proton balance, and Raman spectroscopic studies. Kinetic studies show that compound I formation is base-catalyzed (72). The pKa values are in the range of 3 to 6. The reactions of compounds I and II with substrates are also pH-dependent with pKa values in a similar range (72). Ligand binding (e.g. CO, O2 or halide ions) to ferrous and ferric peroxidases is also pH-dependent. A wide range of pKa values has been reported (72). The redox potentials of Fe3+/Fe2+ couples for peroxidases measured so far are all affected by pH. The pKa values are between 6 and 8, indicative of an imidazole group of a histidine residue (6, 31-33),... 

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. 

Interestingly, the heterocyclic side-chain of histidine is partially ionized at pH 7.0. This follows from... 

We shall see that this modest level of ionization is particularly relevant in some enzymic reactions where histidine residues play an important role (see Section 13.4.1). Note, however, that when histidine is bound in a protein structure, pATa values for the imidazole ring vary somewhat in the range 6-7 depending upon the protein, thus affecting the level of ionization. 

The imidazole side-chain of histidine has a value of 6.0, making it a weaker base than the unsubstituted imidazole. This reflects the electron-withdrawing inductive effect of the amino group, or, more correctly the ammonium ion, since amino acids at pH values around neutrality exist as doubly charged zwitterionic forms (see Box 4.7). Using the Henderson-Hasselbalch equation, this translates to approximately 9% ionization of the heterocyclic side-chain of histidine at pH 7 (see Box 4.7). In proteins, plCa values for histidine side-chains are estimated to be in range 6-7, so that the level of ionization will, therefore, be somewhere between 9 and 50%, depending upon the protein. 

The amino acids in question are the basic amino acids lysine, arginine, and histidine, and the acidic amino acids aspartic acid and glutamic acid. The side-chain functions of these amino acids, ionized at pH 7 (see Box 4.7), act as acids or bases. In a reverse sequence, protons may be acquired or donated to regenerate the conjugate acids and conjugate bases. 

The active site of the enzyme contains a glutamic acid residue that is ionized at pH 7 and supplies the base. A histidine residue, partially protonated at pH 7, in turn supplies the proton necessary to form the common enol (Figure 13.6). 

The process continues, in that the now uncharged histidine is suitably placed to remove a proton from the second of the two hydroxyls, and tautomerization is achieved by abstraction of a proton from the now non-ionized... 

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