Tyrosine residues chemical modification

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

There is only one tyrosine residue in some sea snake neurotoxins. This residue is usually quite difficult to modify, but once it is modified, the toxicity is lost (9). Histidine seems not to be essential as the chemical modification of this residue does not affect the toxicity 10). 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

Other PTMs may involve changes in the chemical nature of amino acids (e.g., citrullination or deimination). Because many of these modifications result in mass changes that are measurable by MS, they are amenable to detection by MS-based approaches. A number of emerging MS-based strategies allow the identification of PTMs. Several MS-based methods to determine the types and sites of protein phosphorylation and ubiquitination have been developed. Phosphorylation occurs mainly on serine, threonine, and tyrosine residues at a frequency ratio of 1800 200 1 in vertebrates.70 Although the phosphorylation of tyrosine residues occurs less frequently in the proteome, it has been extensively studied. 

In 1979, Ross et al 22i" measured the ODMR of tyrosine in glucagon and the derivative [12-homoarginine]glucagon to examine the effect of chemical modification of a lysine residue adjacent to Tyr-10 and Tyr-13. The guanidinated analogue had lower potency than glucagon in a fat cell hormone receptor assay. Since the tyrosine ODMR and other spectral properties of the polypeptide, including circular dichroism, were essentially identical, it was... 

Janing, G. R., Kraft, R., Blanck, J., Rabe, H., and Ruckpaul, K. (1987). Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues. Biochim. Biophys. Acta. 916, 512-523. 

The assumption is an all-or-none normalization of each of the 3 residues. It is interesting how well this appears to explain the data although there is no a priori reason why that should be so. The residues initially labeled A, B, and C have been tentatively identified as Tyr 25, 92, and 97, respectively (300). The normalization of Tyr 92 (B) causes a decrease in molar absorbance at 287 nm of 700 with little change in either viscosity or rotation. Of the three this is the most accessible residue in the X-ray structure. The normalization of the other two causes changes of 1000 each in absorbance and is accompanied by both viscosity and rotation changes. The second to normalize is Tyr 25 (A) presumably by dissociation of the N-terminal portion of the chain from the body of the molecule, which would expose this residue as in S-protein. The last is Tyr 97 (C) whose exposure requires disruption of the entire structure. This residue is also the most buried of all the tyrosine residues according to the X-ray structure. However, the accessibility of Tyr 97 to chemical modification in S-protein and Met 30 to alkylation in RNase-A indicate that the region around Tyr 97 may be easily deformable. If this is so, why should Tyr 97 be the last to normalize. 

The role of certain residues in the enzyme mechanism has been confirmed by chemical modification studies, notably for tyrosine. 14 Modification of tyrosyl residues (for example acetylation or nitration) leads to loss of peptidase activity and enhancement of esterase activity. The presence of the inhibitor -phenylpropionate protects two tyrosine residues from acetylation. Those are Tyr-248 and probably Tyr-198, which is also in the general area of the active site. The modified apoenzyme has lower affinity for dipeptides, as might be expected from the loss of hydrogen bonding between Tyr-248 and the peptide NH group. 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

The mechanisms of modification are different and depend on the chemical molecules used for the reactions. The anhydride of succinic acid reacts with lysine residues, a-amino groups of proteins and free thiol groups. During succinylation, proteins lose their globular structure and undergo aggregation by disulfide cross-binding with other whey proteins. Acetyl anhydride can modify tyrosine residues. 

Reversible chemical modification of enzymes, which was discovered in 1955 by Edmond Fischer and Edwin Krebs [58], is a more prevalent mechanism for cellular signaling switching. Fischer and Krebs showed that enzymes can be turned from an inactive form to an active form via phosphorylation of certain residues of the protein. Enzymes that catalyze phosphorylation (addition of a phosphate group coupled with ATP or GTP hydrolysis) are called protein kinases. Enzymes that catalyze dephosphorylation (which is not the reverse reaction of the phosphorylation) are called phosphatases. For example, a protein tyrosine phosphatase is an enzyme that catalyzes the removal of a phosphate group from a tyrosine residue in a phosphorylated protein [57],... 

Although the chemical modification of tyrosine residues has enjoyed a long history, this residue remains an underused target for bioconjugation reactions. It is typically modified through electrophilic aromatic substitutions (EAS), which makes its reactivity distinct from other amino acid side chains. This reaction... 

Figure 5 Chemical modification strategies for tyrosine residues.

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. 

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