Tyrosine residues normalization

After 1 min exposure to gjjd lactoperoxidase the ratios of radioactivity incorporated per tyrosine residue normalized... 

RPTK activation. The activity of RPTKs is normally suppressed in their quiescent state. This suppression is due to the numerous loose and unstructured conformations of the activation loop (A loop) within the catalytic domain the majority of these conformations interfere with substrate and ATP binding. However, a subset of these conformations is amenable to binding of substrate and ATP, resulting in activation of the RPTKs. Phosphorylation of the tyrosine residue(s) in the A loop shifts the equilibrium towards the active conformation. Because of steric hindrance, PTK catalytic domains appear to be unable to autophosphorylate tyrosine residue(s) in the A loop within the same molecule rather frans-autophosphoryla-tion between two different catalytic domains is necessary for their activation. As a consequence, ligand-induced dimerization is an important step in the activation of RPTKs (Fig. 24-7). 

Ribonuclease is an enzyme with 124 amino acids. Its function is to cleave ribonucleic acid (RNA) into small fragments. A solution containing pure protein, with no other ions present except H+ and OH- derived from the protein and water, is said to be isoionic. From this point near pH 9.6 in the graph, the protein can be titrated with acid or base. Of the 124 amino acids, 16 can be protonated by acid and 20 can lose protons to added base. From the shape of the titration curve, it is possible to deduce the approximate pATa for each titratable group.1-2 This information provides insight into the environment of that amino acid in the protein. In ribonuclease, three tyrosine residues have "normal values of pATa(=10) (Table 10-1) and three others have pA a >12. The interpretation is that three tyrosine groups are accessible to OH, and three are buried inside the protein where they cannot be easily titrated. The solid line in the illustration is calculated from pA"a values for all titratable groups. 

Dye-sensitized photooxidation of methionine occurs selectively in strong formic or acetic acid media (131). All four methionine residues in RNase-A appear to be converted to sulfoxides under these conditions. The product still showed 13% of the initial activity in the Kunitz RNA assay at pH 5 [see, however, Neumann et al. (ISO)]. Apparently an oxygen atom can be accommodated next to each Met sulfur atom in a structure closely resembling that of the native enzyme. The 3 buried tyrosine residues appeared to have been largely normalized. It would be interesting to know if the binding of inhibitors returned these to the buried condition. 

Part 2 again relies on a battery of comparative tests to establish the correspondence of the reduced-reoxidized product and the native enzyme (192). Chromatographic behavior, enzymic activity toward RNA, C > p U > p, UV spectra, ORD, viscosity, and reaction to antisera to RNase-A were all identical with or very similar to those of the starting enzyme, while the reduced material was markedly altered in all of these properties for which tests were possible. It was possible to crystallize the reoxidized material. The crystals were identical in space group, lattice constants, and general intensity distribution of the X-ray reflections within the normal limits of variation to the crystals of RNase-A from the same solvent (193). Recent circular dichroism studies show small differences near 240 nm in reoxidized material (194, 19 ) interpreted as arising from change in the chirality of one disulfide or the environment of one tyrosine residue. 

Ribonuclease contains no tryptophan. The absorption near 280 nm is almost entirely resulting from the 6 tyrosine residues. The ionization of tyrosine produces a marked shift to longer wavelengths in the absorption spectrum. The ionization can be monitored near 295 nm. Shugar (293) was the first to point out the abnormal behavior of 3 of the tyrosine residues on alkaline titration. Three titrate normally with apparent pK values near 10, but three do not titrate until much more alkaline pH values have been reached and irreversible alkaline de-naturation has set in. Some typical spectra and difference spectra are... 

Most studies have concentrated on those conditions where reversible transitions can be demonstrated. However, at neutral pH the thermal transition temperature is high enough to introduce difficulties. Ribo-nuclease kept at 95° at pH 7 for 20 min is irreversibly denatured both in its spectral properties and enzymic activity (337). Tramer and Shugar showed that RNase inactivated at pH 7.8 by heating for 30 min has normalized all of its tyrosine residues as far as alkaline spectrophoto-metric titration is concerned. However, the magnitude of the acid difference spectrum is unaffected although the midpoint has shifted from pH 2 to 3. 

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. 

In normal Hb, the v(Fe—N) of the proximal histidine (F8) is near 220 cm-1 (6). In mutant Hb such as Hb M Iwate and Hb M Boston, F8 histidine and E7 histidine are replaced by tyrosine residues, respectively. In five-coordinate ferric a subunits of these compounds, the v(Fe—O- (phenolate)) bands are observed at 589 and 603 cm-1, respectively (7). 

IRadical cofactors in biological systems have become a subject of increasing interest in recent years (1-3). Tyrosine-based radicals, in particular, have now been identified in several enzymes (4). The tyrosine residue functions as a redox-active cofactor by interconverting between the oxidized phenoxyl radical and the normal phenol or phenolate states. More commonly known redox-active cofactors include transition metal ions, and a few enzymes use both tyrosine residues and metals as partners in effecting redox chemistry. 

The tyrosines of bovine pancreatic ribonuclease (RNase) appear to be a case in point. Three of the total of six tyrosine residues per RNase molecule titrate reversibly with a normal pK of about 10, but the other three titrate only at much higher pH and then irreversibly (Shugar, 1952 Tanford et al., 1955a). These results suggest that the RNase molecule has to undergo a profound structural rearrangement before the three anomalous tyrosines become accessible to titration. Furthermore, the absorption of RNase due to tyrosine residues at about 280 m/i exhibits a hyperchromic effect presumably as a result of the special environment of the three anomalous tyrosines. 

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. 

A spectrophotometric titration of the phenolic groups of myosin and its subunits has been reported by Stracher (1960). The data resemble those shown for ribonuclease in Fig. 11. About two-thirds of the tyrosine residues are titrated normally, and about one-third appear inaccessible in native myosin. An interesting feature is that 6 M urea has no effect at all on the titration curve. 

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