Lysozyme tyrosine

Enzymes modified with N -carbonyldiimidazole (CDI) include horseradish peroxidase 761 /1-lactamase after nitration and reduction,[771 lysozyme, and urease.[781 Ref. [77] describes how the tyrosine side chain of a protein was nitrated, reduced with dithionite to an amino group, and then treated with CDI or A/-(2,2,2-trifluoro-ethoxycarbonyl)imidazole to give the benzoxazolinonyl alanine moiety ... 

Some unnatural amino acids have been designed with this metal-chelating property in mind. For instance, bipyridylalanine (BpyAla, 27) has the bipyridyl group that chelates most transition metal ions and has been successfully incorporated into proteins in E. coli BpyAla was shown to reversibly bind copper ions when incorporated into T4 lysozyme, but a tyrosine in the same location was unable to bind copper, indicating that BpyAla is useful to coordinate copper ions to a protein of interest. 

Liii and Liv absorb at 280 nm, whereas has no tryptophan or tyrosine residues and shows no absorption at 280 nm, but can be detected with ninhydrin after alkaline hydrolyses. This result reveals that ozone causes the oxidation of the methionine residue 105 of lysozyme. 

There is a third region of a protein that is neither on the surface nor in the interior but that is in a cleft. Such regions are often associated with enzyme action and examples show they have (1) intermediate mobility (e.g., tryptophan 62 of lysozyme or the tyrosine of carboxypeptidase) and (2) unfavorable energetics of exposed groups—the entatic state. 

Lysozyme provides one example. The motion of flipping by tyrosine or phenylalanine rings requires an oscillation of the groups of the protein around the aromatic ring. These movements if concerted could be quite small (e.g., —1.0 A). The lysines on the surface must sweep out large conical volumes (e.g., of a base some 5 to 10 A across). The tryptophan motions are small and could be oscillations of 10 to 30°. Valine rotations require little space and methyl rotations almost none. 

Tphe complexing of virtually all purines with aromatic molecules seems - to have far-reaching biological significance. For example, it is known that caffeine affects the rates of many enzymatic reactions (e.g., 0.01, 0.05, and 0.10M caffeine will inhibit salivary amylase 29, 54, and 72% respectively) (12), and purine can decrease the helix-coil transition temperature of the proteins bovine serum albumin and lysozyme (2). It is not unreasonable to expect the involvement of caffeine-aromatic and purine-aromatic complexes because caffeine derivatives and purine complex with the aromatic amino acids tyrosine, phenylalanine, and tryptophan (2). (In fact tryptophan forms a stable 1 to 1 crystalline complex in 0.5M theophylline glycol.)... 

Three tyrosines react with cyanogen fluoride in the neutral range (Gobrinoff 1967). While all four residues react with the tetranitromethane, only two are nitrated (Habeeb and Atassi 1971 Denton and Ebner 1971). These observations are generally consistent with the proposed model (Warme et al 1974). The disulfide bonds in a-lactal-bumin, as predicted from the expanded model, are more rapidly reduced and, therefore, more accessible than in lysozyme (Iyer and Klee... 

Some studies have been reported where individual aromatic groups of a protein have been systematically replaced using site-directed mutagenesis, allowing the contributions of the individual chomophores to be determined. Craig et al. [106] have systematically relaced all the Trp moieties in interleukin-ip (IL-lp). Earlier work of Elwell and Schellman [107] replaced the tryptophan in T4 lysozyme with tyrosine. 

With several other proteins, such as bovine serum albiunin (Tanford and Roberts, 1952), lysozyme (Tanford and Wagner, 1954), and/3-lacto-globulin (Tanford and Swanson, 1957), pK shifts of the phenolic OH groups of tyrosine residues are observed, but these are of a qualitatively different nature. Thus, the tyrosines of any one of these proteins cannot be readily differentiated into a normal and an abnormal variety, since the spectrophotometric titration data for these proteins are reversible and fall on single smooth curves, in contrast to the situation with RNase. On the other hand, the tyrosine residues of ovalbumin show comparable behavior to the three abnormal tyrosine groups of RNase (Crammer and Neuberger, 1943). About 2 of the total of 9 tyrosine residues appear to titrate normally, but the remainder are not titrated up to pH 12. At pH 13, these anomalous tyrosines become titratable, and this is accompanied by the irreversible denaturation of the ovalbumin molecule. 

Luse and M(iLaren (1963) have reviewed published research on the photolysis products and quantum yields tor the destruction of amino acids and have attributed the photochemical inactivation of the enzymes chymo-trypsin, lysozyme, ribonuclease, and trypsin by UV light at 254 m i primarily to destruction of the cystyl and tryptophyl residues. The destruction of these residues in proteins was suggested to be a function of the product of the number of residues present, the molecular extinction coefficient, and the quantum yield for destruction of each residue. Cysteine and tryptamine were identified among the irradiation products from cystine and tryptophan, respectively. Tyrosine, histidine, and phenylalanine were also shown to be degraded by UV, histidine yielding histamine, urocanic acid, and other imidazole derivatives, and phenylalanine yielding tyrosine and dihydroxyphenylalanine. Destruction of these three amino acids was not considered to contribute appreciably to the enzyme inactivation. 

Proton resonance spectra of denatured proteins consist of sharp peaks which correspond to a summation of resonances from individual residues [129]. In C spectra of denatured proteins, it is possible to distinguish all the carbon resonances of the aromatic side chains of histidine, phenylalanine, tyrosine and tryptophan, and separate resonances from alanine, arginine, glycine, isoleucine, leucine, threonine, valine and occasionally methionine [130]. (A natural abundance C spectrum of a 13 mM solution of lysozyme takes only 4 hr accumulation time using 20 mm sample tubes [131]). 

FIGURE 13.12. Electron density for tyrosine 62 in human leukemic lysozyme. Two different conformations of the side chain fit the electron-density map, indicating disorder. (Courtesy D. C. Phillips)... 

Santus R., Patterson L.K., Hug G.L., Bazin M., Maziere J.C., Morliere P., Interactions of superoxide anion with enzyme radicals kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer. Free Rad. Res., 2000,33,383-391. 

Fig. 20. Primary structure of hen-egg lysozyme. ALA alanine, ARG arginine, ASN asparagine, ASP aspartic acid, CYS cysteine, GLN glutamine, GLU glutamic acid, GLY glycine, HIS histidine, ILE isoleucine, LEU leucine, LYS lysine, MET methionine, PHE phenylalanine, PRO proline, SER serine, THR threonine, TRP tryptophan, TYR tyrosine, VAL valine. (Redrawn from Canfield and Lu, 1965).

The effect of nonenhancers has been exploited in an assay for tyrosine in which the attenuation of luminol oxidation enhanced by p-iodophenol was proportional to the concentration of added tyrosine (C8). As a validation of the method, the number of tyrosines in lysozyme was found to be 3, which is in accord with its known amino acid sequence. 

---