Tyrosine ionizing groups

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. 

Riiterjans and Witzel (280) have carefully measured the chemical shift of the C2 proton of the His residues as a function of pH at low ionic strength. The data for His 12 and 119 cannot be fit by ionization curves for simple monobasic acid. The curves are clearly biphasic and indicate a close coupling of two ionizable groups with similar pK values. The authors site this as evidence for a direct interaction between the two imidazole rings but this is not necessary. The phenolic and amino groups of tyrosine show such ionization coupling. These authors also... 

A new motif for OP binding to tyrosine has been identified. Almost all proteins appear to be capable of binding OP covalently on tyrosine. Whether or not OP will bind to tyrosine in vivo will depend on the concentration of the protein, the concentration of the OP, and the ionization status of the tyrosine hydroxyl group. The latter factor appears to be dependent on the presence of nearby positively charged residues. 

Fig. 10. The eight microscopic forms of tyrosine and the twelve ionization constants which interrelate them. Parentheses enclose symbols indicating the charge on each of the ionizing groups, in the sequence carboxyl, phenolic hydroxyl, amino. (Martin el al., 1958 redrawn with permission.)...

Figure 3. L-Tyrosine (a) groups shown as un-ionized with pK values (b) zwitterionic form, typical at moderate pH levels (c) single anion form, e.g., at pH 10 (idealized case with no hydroxyl group ionization).

Tyrosine (CgHi 1NO3), another amino acid in proteins, has two dominant ionizable groups with P HA values of 2.24 and 9.04 at 25°C (and a third, less easily ionizable group with a pAT value of 10.10, which we will neglect here). Its water solubility at this temperature is 0.46 g/kg of water. y... 

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... 

In studies of the recombination of heme with globin Jope,. lope and O Brien (1949) have used the concept of bound tyrosine hydroxyl groups as a criterion of native character in globin, based on the spectropho-tometric study of the alkaline ionization process. They were able to show that globin preparations which were native according to this criterion could be classified as denatured on the basis of the position of their tryptophan fine-structure bands when recombined with heme. These results led them to suggest that the process of denaturation could be separated into several stages, even by spectroscopic techniques alone (see also Jope, 1949). 

For human serum albumin Tanford (1950) found by spectrophotometry that the ionization of the tyrosine hydroxyl groups was completely reversible up to pH 12. Measurements at the wavelength of the tyrosine anion maximum (2930 A.), uncorrected for the small tryptophan contribution, gave a pK of 11.7 for this process. Both the ultraviolet absorption and titration data for this protein could be quantitatively interpreted on the basis of complete freedom of all the 18 tyrosine hydroxyl groups in the molecule to ionize. In this respect human serum albumin thus resembles insulin and not ovalbumin. 

This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy-group of L-tyrosine-5 01. The variation of Q for Mg -free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg as well as L-tyrosine-501. 

In a zwitterion, each ionizing group has two constants, one for the fraction that has the other group ionized and one for the remaining fraction. These are called microscopic constants, and the macroscopic constants (as determined by spectrometry or potentiometric titration) are gross composites of these. Historically, tyrosine was one of the first zwitterions to be examined for microconstants (Edsall, Martin and Hollingworth, 1958) and the same methods are still used. See Section 12.4 for the pursuit of microconstants in the catecholamine series. 

Fig. 153. The pH dependence of the difference spectrum of 0.24% (1.6 X 10 M) solutions of lysozyme at 0.15 ionic strength. Reference pH s are approximately 1.2. Open circles,25 C. filled squares, 1 C. Open squares, the pH dependence of the tyrosine ionization in the methylated lysozyme. Dashed curve is a calculated difference spectrum for tyrosine, with p i t 10.80 for the three groups, and 0.080 for w. The solid curves are calculated difference spectra for the following groups 1 group with pKi t 4.20, = 0, AD = 0.070 1 group with pjTint 6.85, Aff = 3 kcal., AD = 0.060 3 groups with pKint 10.80, AD = 6 kcal., AD s= 1.10...

Although it has long been accepted that the emission from proteins in the 350 nm region is largely attributable to tryptc han, recent work by Rayner et al. has raised the possibility that excited state ionization of the tyrosine phenolic groups occurs in proteins under neutral pH conditions and thus tyrosinate emission may significantly contribute to protein fluorescence. 

---