Methionine residues modification

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Vithayathil, P.J., and Richards, F.M. (1960) Modification of the methionine residue in the peptide component of ribonuclease-S./. Biol. Chem. 235, 2343-2351. 

The sulfur atom of methionine residues may be modified by formation of sulfonium salts or by oxidation to sulfoxides or the sulfone. The cyanosulfonium salt is not particularly useful for chemical modification studies because of the tendency for cyclization and chain cleavage (129). This fact, of course, makes it very useful in sequence work. Normally, the methionine residues of RNase can only be modified after denaturation of the protein, i.e., in acid pH, urea, detergents, etc. On treatment with iodoacetate or hydrogen peroxide, derivatives with more than one sulfonium or sulfoxide group did not form active enzymes on removal of the denaturing agent (130) [see, however, Jori et al. (131)]. There was an indication of some active monosubstituted derivatives (130, 132). 

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 ... 

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. 

Cigarette smoke decreases the < i-PI activity in rat lung and produces a functional deficiency of protease inhibitors in the lower respiratory tract of humans (11,12). It is quite reasonable to assume that this is due to the oxidation of the essential methionine residue in i-PI. Thus, chemical modification of ax-PI by oxidation of a methionine residue to a methionine sulfoxide residue by some component of cigarette smoke or by myeloperoxidase which is released by cigarette smoke, results in inactivation of this essential protease inhibitor. The resulting imbalance of proteases and protease inhibitors in the lung then results in the development of emphysema. 

The same approach was used to study the reductive modification of a methionine residue (Met ) in the amyloid-/ peptide [A/ (l-40)] and its reversed sequence [A/ (40-l)]. The A/3 peptide suffers the highly selective attack of H atoms on the Met residue, with the formation of a modified peptide containing an a-amino-butyric acid residue. Formation of tw -lipids in POPC system as a marker of radical damage to A/3 peptide clearly shows the transfer of radical damage from the peptide to the lipid domain. 

Kadlcik V, Sicard-Roselli C, Houee-Levin C, Kodicek M, Ferreri C, Chatgilialoglu C. (2006) Reductive modification of a methionine residue in the amyloid-j8 peptide. Angew Chem IntPd 45 2595-2598. 

These derivatizations are highly selective, and may thus allow PSD measurements to be carried out on peptides after modification. Such a protocol would significantly enhance our ability to derive sequence information from PSD spectra, because the mass shifts observed in fragments help locate the particular residue within the peptide, and also confirm assignments of fragments as arising from N- or C-terminal regions. In addition to derivatizations that may modify the C- and N-termini and the derivatization of tyrosine residues, we have carried out oxidation of methionine residues with sufficient specificity to enable measurement of PSD spectra. 

Modification by performic acid oxidation Treatment of proteins with performic acid leads to the oxidation of cysteine and cystine residues to cysteic acid residues (Sanger 1949). Methionine residues are quantitatively converted to the sulfone (Hirs 1956), and tryptophan undergoes oxidative destruction (Toennies and Homiller 1942 Benassi et al. 1965). Other amino acids are not modified, provided that precautions are taken to avoid chlorination (Thompson 1954 Hirs 1956), or bromination (Sanger and Thompson 1963) of tyrosine residues. Cleavage of peptide bonds does not occur on performic acid oxidation at low temperature. 

The degradation product of the sulfonimn salt formed in the greatest yield after acid hydrolysis is undoubtedly a function of the structure of the affinity label. Generally, if the total amino acid composition of the modified protein was determined, low yields of methionine and increased yield of homoserine would be indicative, but certainly not proof of, the modification of a methionine residue. Sulfonium salts of methionine are generally not oxidized by hydrogen peroxide (Sigman and Blout 1967). 

It is worth noting that the radical damage to methionine-containing peptides and proteins consists of a desulfurization process, which leads to the replacement of a methionine residue with an a-aminobutyric acid in the sequence. This could be a posttranslational modification, which is linked to a postsynthetic modification of lipids by the above-reported tandem mechanism. A chemical biology approach can be proposed involving lipidomics and proteomics, in order to configure the metabolic changes related to a radical stress. 

Another possible mechanism for the activation of the enzyme is shown in Figure 6-8B. This model designated as Model II, takes into consideration that in the amino acid sequence of the enzyme there is a methionine residue that is conserved in position 129. In this model, HP heme reduction (the C-terminal domain heme) promotes a drastic modification of the coordination sphere of LP heme (the N-terminal domain heme) due to the replacement of the axial His 85 by Met 129. The intro-... 

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