Tyrosine residues, modification

Protein tyrosine kinases (PTKs) are enzymes (EC 2.7.1.112) that catalyze the transfer of the y-phosphate group of ATP to tyrosine residues of protein substrates. The activity of PTKs is controlled in a complex manner by posttranslational modifications and by inter- and intramolecular complex formations. 

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). 

Originally, for preparation of such conjugates the hydroxyl groups of monomethoxy-PEG (mPEG) were activated with cyanuric chloride, and the resulting compound then coupled with proteins (10). This approach suffers from disadvantages, such as the toxicity of cyanuric chloride and its limited applicability for modification of proteins having essential cysteine or tyrosine residues, as manifested by their loss of activity. 

It has been already pointed out that nitric oxide exhibits antioxidant effect in LDL oxidation at the NO/ 02 ratio 1. Under these conditions the antioxidant effect of NO prevails on the prooxidant effect of peroxynitrite. Although some earlier studies suggested the possibility of NO-mediated LDL oxidation [152,153], these findings were not confirmed [154]. On the other hand, at lower values of N0/02 ratio the formed peroxynitrite becomes an efficient initiator of LDL modification. Beckman et al. [155] suggested that peroxynitrite rapidly reacts with tyrosine residues to form 3-nitrotyrosine. Later on, Leeuwenburgh et al. [156] found that 3-nitrotyrosine was formed in the reaction of peroxynitrite with LDL. The level of 3-nitrotyrosine sharply differed for healthy subjects and patients with cardiovascular diseases LDL isolated from the plasma of healthy subjects contained a very low level of 3-nitrotyrosine (9 + 7 pmol/mol 1 of tyrosine), while LDL isolated from aortic atherosclerotic intima had a 90-fold higher level (840 + 140 pmol/moD1 of tyrosine). It has been proposed that peroxynitrite formed in the human artery wall is able to promote LDL oxidation in vivo. 

The covalent modification of cellular proteins by phosphorylation of serine/ threonine and tyrosine residues provides an efficient molecular switch for altering cellular responses. 

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. 

This enzyme removes C-terminal tyrosine residues from the a subunits of tubulin (Hal-lak et a/., 1977 Argarana et aL, 1978 Martensen, 1982). The role of this modification reaction remains to be established... 

Protein sulfation occurs exclusively at tyrosine residues." It has been suggested that up to 1 % of the tyrosine protein content becomes sulfated, which is the most abundant posttranslational modification for tyrosine, with phosphorylation occurring only on 0.5% of tyrosine protein content." Sulfation occurs mostly on excreted proteins or trans-membrane proteins. Sulfation is catalyzed by tyrosylprotein sulfotransferase (TPST), with PAPS as a cosubstrate (Scheme 4). Like kinases, sulfotransferases have a biological inverse known as sulfatases." ... 

Processing of a newly synthesized type V procollagen molecule is also different from that of type I collagen. BMP-1, which is the C-propeptidase of type I, II, and III collagens, cleaves the N-terminal propeptide of the pro-al(V) chain, and a furin-like proteinase cleaves the C-terminal propeptide. The tyrosine residue in the N-terminal NC domain of type V collagen is sulfated. This modification might be related to... 

Deleterious protein cross-linking can also be induced by reactive nitrogen species (RNS) such as peroxynitrite ONOO formed by the reaction of superoxide with nitric oxide (NO). The cross-links are formed between tyrosine residues following nitration by peroxynitrite (Sitte, 2003). Carnosine appears to play roles not only in NO generation but also in protection against excess NO production by inducible nitric oxide synthetase (NOS), thereby preventing ONOO-mediated protein modification (Fontana et ah, 2002). Evidence for a carnosine-NO adduct has also been published (Nicoletti et al., 2007). 

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. 

Many enzymes may be regulated by covalent modification, most frequently by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. 

Many enzymes are regulated by covalent modification, most frequently by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. In the fed state, most of the enzymes regulated by covalent modification are in Ihe dephosphorylated form and are active (see Figure 24.2). Three exceptions are glycogen phosphorylase (see p. 129), fructose bis-phosphate phosphatase-2 (see p. 98), and hormone-sensitive lipase of adipose tissue (see p. 187), which are inactive in their dephosphorylated state. 

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. 

Confirmation of this possibility comes from the work of Steinberg and Sperling. From the completely reduced protein they have produced a derivative containing 1 mercury atom bridging each of the 4 pairs of sulfur atoms 188). The resulting molecule had two abnormal tyrosine residues and reacted with antiserum to RNase. In a more limited modification 1 mercury atom was introduced specifically at the 65-72 SS group 184). This derivative was fully active and nearly identical to RNase-A. 

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. 

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