Iodoacetate methionine residues

Ribosomal protein L12 was oxidized with 0.3 M H202 at 30°C for 1 h. After dialysis, the protein was incubated in the presence of 0.8 M 2-mercaptoethanol for 48 min at 37 °C and dialyzed. The amount of methionine residues was quantitated by exhaustive alkylation of the protein with [14C]iodoacetic acid. 

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 investigations of W. H. Stein and Moore and their colleagues were first reported in 1959 157). The inactivation of RNase by iodo-acetate was studied. A maximum in the rate of activity loss was noted at pH 5.5. Reaction with a methionine residue was found at pH 2.8 at pH 8.5-10 lysine residues were modified, but at pH 5.5-6.0 only histidine appeared to be involved. The specific reaction required the structure of the native enzyme. Reaction with histidine was not observed under a variety of denaturing conditions 158). Iodoacetamide did not cause activity loss, or only very slow loss, or alkylate His 119 in the native enzyme at pH 5.5. The negative charge on the carboxyl group of the iodoacetate ion was apparently essential. 

Methionine 13 in S-peptide has been modified by oxidation to the sul-fone or by conversion to a sulfonium salt with either iodoacetic acid or iodoacetamide (138). There is a dramatic lowering of the peptide-protein binding constant for all of the derivatives, but the complexes when formed appear to have nearly normal catalytic activity. The X-ray structure does not appear to permit the normal sulfur location with the sulfonium salts. Sterically, Met 13 can be moved by rotation about the carbon a-carbon / bond so that the residue sticks out into the solvent. This can be done without any major change in the conformation of the rest of the peptide. Thus the active site could be maintained undisturbed while the contribution of Met 13 to the S-peptide S-protein association would be lacking. 

When RNase-S was treated with iodoacetate at pH 6, both inactivation and histidine modification occurred 164). The modified histidine was in S-protein and was assumed to be His 119 since the sole product on analysis was 1-CM-His. In the absence of S-peptide only methionine modification occurred in S-protein. The loss of potential activity probably resulted from the reaction of the second of the two modifiable Met residues. The location of these residues in the sequence was not established. 

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