Tyrosine tetranitromethane

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... 

The catalytically essential nature of tyrosine 85 and its proximity to the substrate binding site and to tyrosine 115 were demonstrated from studies of modification with tetranitromethane (71) and from studies of intramolecular cross-linking of aminotyrosyl residues (72). The bro-moacetamidophenyl (69) and diazonium (70) reagents obtained from aminophenyl-pdT both react selectively and exclusively with tyrosine 85. This residue is situated, stereochemically, such that its hydroxyl group can interact with the 3 -phosphate of pdTp. 

Various experimental evidence suggests that only 2 or 3 of the 9 tyrosine residues are on the surface of the enzyme (19, 55). Indeed only a part of the tyrosine residues can be easily modified by acetylimidazole at pH 7.5 or by tetranitromethane at pH 8.0 (H. Kasai, K. Takahashi, and T. Ando, unpublished). As enzymes thus modified have catalytic activity, the tyrosine residues that are probably located at the surface of the enzyme do not seem to be essential for activity. Consistent results were also obtained from the modification by fluorodinitrobenzene or by diazo-lH-tetrazole (H. Kasai, K. Takahashi, and T. Ando, unpublished). Especially noteworthy is the derivative, in which one to two tyrosine residues, amino terminal alanine, and one lysine residue were modified with diazo-lH-tetrazole. The derivative was deprived of most of its activity toward RNA but retained about 50% of its activity toward guanosine 2, 3 -cyclic phosphate. This may be explained by some steric hindrance owing to the modification of a tyrosine residue near the active center. 

The presence of an essential tyrosine residue near the active site was suggested on the basis of experiments with tetranitromethane.54 Treatment of apotryptophanase with this reagent caused almost complete loss of catalytic activity, a great reduction of affinity for PLP and modification of about one tyrosine residue. The modified enzyme was unable to form the quinonoid intermediate with L-tryptophan or L-alanine.55 PLP protected the apoenzyme from inactivation only in the presence of activating cations (K+, NH4+, Rb+). It was shown that inactivation by tetranitromethane was not caused by oxidation of SH-groups, but partial modification of methionine (0.8 residue) was detected and might also be responsible for inactivation. It is worthy of note that modification of tryptophanase with chloramine T indicated that some methionine residues may be important for maintaining the catalytically active conformation of the enzyme.56 ... 

Tyrosine is more fluorescent than tryptophan in solution, but when present in proteins, its fluorescence is weaker. This can be explained by the fact that the protein tertiary structure inhibits tryosine fluorescence. Also, energy transfer from tyrosines to tryptophan residues occurs in proteins inducing a total or important quenching of tyrosine fluorescence. This tyrosine — tryptophan energy transfer can be evidenced by nitration of tyrosine residues with tetranitromethane (TNM), a highly potent pulmonary carcinogen. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on the phosphorylation and dephosphorylation of tyrosine, and the subsequent effects on cell proliferation, can be investigated. 

Nitration of phenolic compounds leads to the formation of 3-nitrotyrosine (molar extinction coefficient = 14 400 at 428 nm) (Figure 7.11). The reaction is very specific for phenolic compounds. Chemical nitration of functionally important tyrosine residues by tetranitromethane has often been found to inactivate or alter the enzyme properties. It was only after the detection of in vivo nitrotyrosine formation under inflammatory conditions that the physiological aspects of nitrotyrosine metabolism came to light. Abundant production (1-120 /uM) of nitrotyrosine has been recorded under a number of pathological conditions such as rheumatoid arthritis, liver transplantation, septic shock, and amyotrophic lateral sclerosis (Balabanli etal. 1999). 

Froschle, M., Ulmer, W. and Jany, K.D. (1984). Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state. European Journal of Biochemistry, 142, 533-540. 

Riordan, J.F., Wacker, W.E.C. and Vallee, B.L. (1966). Tetranitromethane. A reagent for the nitration of tyrosine and tyrosyl residues of proteins. Journal of The American Chemical Society, 88, 4104-4105. 

When native PGH synthase was treated with tetranitromethane, three tyrosine residues, Tyr 355, Tyr 385, and Tyr 417, were modified (28). These three tyrosines are possibly important to cyclooxygenase activity. When the native enzyme was first incubated with indomethacin, a known cyclooxygenase inhibitor, none of these tyrosine residues was nitrated. In the same work, site-directed mutagenesis was used to replace each of these tyrosines with phenylalanine, and only the mutant Y385F lacked cyclooxygenase activity. This result suggests that Tyr 385 is located near the site of cyclooxygenase activity and is the source of the tyrosyl radical responsible for H atom abstraction from arachidonic acid. 

It is unlikely that the introduction of the probe itself induces these changes in protein conformation since recent studies with nitrocarboxy-peptidase (64) confirm the findings of Johansen and Vallee (62, 63). Treatment of carboxypeptidase crystals with tetranitromethane exclusively nitrates tyrosine-248. In solution nitrocarboxypeptidase exhibits a visible absorption band with a maximum at 428 nm titrating with a pK of about 6.3. This abnormally low pK value (relative to model nitro-... 

Phenol nitration with tetranitromethane (37) (TNM) is typically run at pH 8 at room temperature and is selective for tyrosine residues under these conditions (although some oxidation of cysteine residues has been reported) (38). The product of this reaction could be thought to develop through an electrophilic... 

Finally, treatment with reagents such as tetranitromethane, may lead to products in which the expected tyrosine derivative is absent. The extent of formation of such derivatives is difficult to assess from amino acid analyses. 

Tetranitromethane is used as an oxidizer in rocket propellants, as a diesel fuel additive, and as an explosive. It is used as a biochemical agent to nitrate tyrosine proteins. It is also used as an organic reagent for detecting the presence of double bonds, and as a mild nitrating reagent, reacting with tyrosine residues in proteins and peptides. 

Kievan and Tse (1983) have examined the effect of chemical modification of E. coli topoisomerase I and DNA gyrase by tetranitromethane, which reacts preferentially with tyrosine residues. With each enzyme, treatment with tetranitromethane led to abolition of the topoisomerase activity. Moreover, the enzymes were protected from this inactivation when bound to DNA, implying that some of the modified residues are involved in DNA binding. However, this study does not identify the tyrosine residues involved in the protein-DNA bond as being the amino acid residues whose modification inactivates the enzyme. In the case of DNA gyrase, which has two subunits, it was not determined which subunit was inactivated. 

Controlled reaction of G. gouldii hemerythrin with tetranitromethane has shown that tyrosyl residues 8 and 109 and, to a lesser extent, tyrosyl 67 are protected from nitration in the iron-protein (201—202). The more readily nitrated residues, namely tyrosines 18 and 70 are, hence, likely to he on the surface of the monomer. This partial modification did not affect either iron-binding, as judged by its visible absorption spectrum,... 

It is also possible to attenuate the enzymic activity by reacting tyrosine with tetranitromethane (210), but no peptide has been isolated. Possibly tyrosine-85 or -246 which are involved in binding of the coenzyme may be relevant to some of these results. 

Price and Radda (338) found that N-acetylimidazole could acetylate up to six tyrosine residues without loss of activity or alteration of Km for substrate however, reaction of about one tyrosine per subunit results in desensitization toward GTP, but the response to ADP is not abolished even by extensive 0-acetylation. Essentially the same results are observed upon nitration with tetranitromethane (TNM). Acetylation does not grossly alter the molecular weight, as measured by sedimentation velocity, or the conformation, as determined by ORD. The GTP site is not protected by NADH alone, but is partially protected (25-50%) by GTP and is at least 75% protected by inclusion of both GTP and NADH in the reaction mixture. Piszkiewicz et al. (339) confirmed these findings by modification with TNM. The reaction is biphasic with initial rapid formation of one residue of 3-nitrotyrosine per subunit. The primary site of reaction is tyrosine-406 in the linear sequence (340). Later (338) the same effect was obtained with chicken GDH with both enzymes there is no influence on activation by ADP. Further, the pH optima of the enzymes are not influenced by the degree of nitration or the inhibition by GTP or activation by ADP (338). 

Figure 4.21 Tyrosine reactive probes. Nitration of tyrosine by reaction with tetranitromethane, followed by reduction with sodium dithionite, to yield an o-aminotyrosine.

Tetranitromethane (TNM) is a highly potent pulmonaty carcinogen. The toxic and carcinr enic mechanism(s) of TNM and related nitro confounds are unknown. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on phosphorylation and dephosphorylation of tyrosine, and subsequent efTeds on cell proliferation can be investigated. 

The modification of all four tyrosine residues (Tyr-399, 408,449, and 494) of fragment 377-571 by nitration with tetranitromethane yielded a homogeneous derivative which, by circular dichroism and optical rotatory dispersion measurements, had suffered no conformational alterations. Disulfide availability studies, however, indicated a slight increase in the reducibility of its disulfide bonds. The nitrated derivative behaved in an identical manner to unmodified peptide 377-571 in precipitin reactions with antisera to 377-571, and was also equally as effective in inhibiting the precipitin reaction of BSA with antisera to BSA. Also, with each of the antisera tested in immunoadsorbent studies, the derivative... 

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