Tyrosyl compounds

Having obtained expressions [Eqs. (II-23) to (II-25)] for the thermodynamic parameters for the ionization of hydrogen-bonded donor groups, we are now in a position to obtain an experimental verification of the values deduced for the thermodynamic parameters for the formation of hydrogen bonds in Section 5. For this purpose we shall make use of data of Tanford et al. (1955b) for the ionization of tyrosyl groups in bovine serum albumin. In the protein AF h. was found to be 14.1 kcal. per mole, compared with the value AF° = 13.1 kcal. per mole observed for model tyrosyl compounds, i.e., the tyrosyl groups in bovine serum albumin are abnormal in that they are weaker acids than would be expected. If these values are substituted into the first of Eqs. (II-23), a value of 4 is obtained for Kij (Loeb and... 

The reaction of peroxynitrite with the biologically ubiquitous C02 is of special interest due to the presence of both compounds in living organisms therefore, we may be confident that this process takes place under in vivo conditions. After the discovery of this reaction in 1995 by Lymar [136], the interaction of peroxynitrite with carbon dioxide and the reactions of the formed adduct nitrosoperoxocarboxylate ONOOCOO has been thoroughly studied. In 1996, Lymar et al. [137] have shown that this adduct is more reactive than peroxynitrite in the reaction with tyrosine, forming similar to peroxynitrite dityrosine and 3-nitrotyrosine. Experimental data were in quantitative agreement with free radical-mediated mechanism yielding tyrosyl and nitric dioxide radicals as intermediates and were inconsistent with electrophilic mechanism. The lifetime of ONOOCOO was estimated as <3 ms, and the rate constant of Reaction (42) k42 = 2 x 103 1 mol 1 s 1. 

Other very convincing evidences for free radical-mediated mechanism of decomposition and reactions of peroxynitrite and nitrosoperoxocarboxylate were demonstrated by Lehnig [140] with the use of CIDNP technique. This technique is based on the effects observed exclusively for the products of free radical reactions their NMR spectra exhibit emission characterizing a radical pathway of their formation. Lehnig has found the enhanced emission in the 15N NMR spectra of N03- formed during the decomposition of both peroxynitrite and nitrosoperoxocarboxylate. This fact indicates that N03- was formed from radical pairs [ N02, H0 ] and [ N02, C03 ]. Emission was also observed in the reaction of both nitrogen compounds with tyrosine supposedly due to the formation of radical pair [ N02, tyrosyl ]. 

The ability of MPO to catalyze the nitration of tyrosine and tyrosyl residues in proteins has been shown in several studies [241-243]. However, nitrite is a relatively poor nitrating agent, as evident from kinetic studies. Burner et al. [244] measured the rate constants for Reactions (24) and (25) (Table 22.2) and found out that although the oxidation of nitrite by Compound I (Reaction (24)) is a relatively rapid process at physiological pH, the oxidation by Compound II is too slow. Nitrite is a poor substrate for MPO, at the same time, is an efficient inhibitor of its chlorination activity by reducing MPO to inactive Complex II [245]. However, the efficiency of MPO-catalyzing nitration sharply increases in the presence of free tyrosine. It has been suggested [245] that in this case the relatively slow Reaction (26) (k26 = 3.2 x 105 1 mol-1 s 1 [246]) is replaced by rapid reactions of Compounds I and II with tyrosine, which accompanied by the rapid recombination of tyrosyl and N02 radicals with a k2i equal to 3 x 1091 mol-1 s-1 [246]. 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

In photosystem II an intermediate tyrosyl radical is formed which then repetitively oxidizes an adjacent manganese cluster leading to a four-electron oxidation of two water molecules to dioxygen. In broad detail, the model compounds" described above were demonstrated to undergo similar reactions on photochemical excitation of the respective ruthenium centers. 

A number of studies on the fluorescence decay of tyrosine, tyrosine derivatives, and small tyrosyl peptides have been carried out. 36-38 Whereas the tyrosine zwitterion and tyrosine derivatives with an ionized a-carboxy group exhibited monoexponential fluorescence decay (x = 3.26-3.76 ns), double- or triple-exponential decay was observed in most other cases. As in the case of the tryptophan model compounds, the complex decay kinetics were again interpreted in terms of rotamer populations resulting from rotation around the C —Cp bond. There is evidence to indicate that the shorter fluorescence lifetimes may arise from rotamers in which the phenol ring is in close contact with a hydrated carbonyl group 36 37 and that a charge-transfer mechanism may be implicated in this quenching process. 39 ... 

Tyrosyl radical Fe(TV)Tyr is formed by an internal electron transfer in the peroxidase Compound I ... 

What happens when the length of the substrate is extended in the direction of the N terminus The tyrosyl residue in the foregoing compound may be designated P,. For extended substrates which contain P,3 and additional residues (this includes most natural substrates) the Km values decrease very little from that of short substrates despite the larger number of "subsites" to which the substrate is bound. However, the maximum velocity is often much greater for the extended substrates than for short ones. Thus, for N-acetyltyrosyl-glycine amide Km is 0.017 M, only a little less than for N-acetyltyrosine amide, but fccat is 7.5 s 1,440 times greater than for the shorter substrate.229 288 289 Other examples have been tabulated by Fersht.279... 

When a 2 -Cl or -F analog of UDP was used in place of the substrate an irreversible side reaction occurred by which Cl or F, inorganic pyrophosphate, and uracil were released 349 When one of these enzyme-activated inhibitors containing 3H in the 3 position was tested, the tritium was shifted to the 2 position with loss of Cl and formation of a reactive 3 -carbonyl compound (Eq. 16-24) that can undergo P elimination at each end to give an unsaturated ketone which inactivates the enzyme. This suggested that the Fe-tyrosyl radical abstracts an electron (through a... 

Several applications of photoreactive peptides require the presence of a radionuclide to allow specific and sensitive detection of the photo-cross-linked conjugates. In several cases, radioiodination of tyrosyl moieties and radiolabeled Bolton-Hunter reagents have been used. However, the presence of a radiolabel within the benzophenone photophore is desirable, particularly when the objective is to identify the site of photo-insertion of benzophenone. To this end some radiolabeled, benzophenone-based compounds have been developed and used in peptide synthesis, in particular tritiated Phe(4-Bz) (Scheme 24)J2161 [1-14C-carboxy]-4-benzoylbenzoic acid,1221 and 4-benzoyl-(2,3-3H2)-dihydrocinnamic acid.[154l In addition, 4-(4-hydroxybenzoyl)phenylalanine (Scheme 25) has been directly radioiodinated with Na125I and Chloramine-T)151 ... 

Aminoacyl adenylates have long been known to be high energy compounds, but their free energies of hydrolysis had not been accurately measured. This was accomplished for tyrosyl adenylate using the Haldane approach (Chapter 3, section H) and mutants of the tyrosyl-tRNA synthetase. The equilibrium constant for the formation of tyrosyl adenylate in solution (Absolution) = [Tyr-AMP] [PPi]/ [Tyr] [ATP]) is related to the rate and equilibrium constants for the enzymatic reaction illustrated in Figure 15.21 by equation 15.8. 

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