Iodoacetate histidine residues

Alkylation at pH 8.5 shows reduced rates of reaction at the histidine residues but significant substitution at lysine, particularly Lys 41 118). The histidine reactions show the same general stereospecificity as found at pH 5.5. The inactive Lys 41 derivatives (25, 26, and 27 of Table VI) show alkylation patterns of His 12 and 119 at pH 5.5 which are similar to those of RNase-A although with some differences in detail. When Lys 1 and 7 are acetylated in RNase-S the alkylation pattern with iodoacetic acid is not affected. When PIR is used the alkylation of His 119 is nearly abolished but that at His 12 is accelerated 163). The probable interaction of Asp 121 with His 119 may be important in the alkylation reactions observed in the native enzyme and the various lysine derivatives. In PIR this interaction has, of course, been removed. 

RNase A is completely inhibited if either of two histidine residues (His 12 or His 119) is modified by car-boxymethylation with iodoacetate (fig. 8.13) suggesting that these histidines play important roles in the active site. In support of this conclusion, the reaction of iodoacetate with His 12 or His 119 is inhibited by cytidine-3 -phosphate and other small molecules that bind at the active site. Lysine 41 has been implicated similarly in the active site by the observation that enzymatic activity is destroyed by the reaction of... 

The presence of imidazole groups in the active site region of human carbonic anhydrase B has, in fact, been demonstrated by chemical modification. Thus, bromoacetate reacts specifically with the 3 -N of a histidine residue to give a partially active monocarboxymethyl enzyme (65). The reaction depends on the initial combination of the bromoacetate ion with the anion binding site (65,83). In a detailed study, Bradbury (83) has shown that the irreversible reaction at saturation with iodoacetate... 

Another potent group of alkylators are the a-haloketones. The classic example is the alkylator TPCK (tosyl-L-phenylalanylchloromethyl ketone) which interacts specifically with one imidazole ring (histidine residue) in the enzyme a-chymotrypsin (see Section 7.2.3). These compounds are significantly more reactive in Sn2 displacements than alkyl halides. For example, nucleophilic attack by iodide will proceed 33,000 times as quickly on chlo-roacetone as on n-propyl chloride (acetone solvent, 50°C). Inductive pull of the carbonyl function would be expected to both increase the electrophilicity of the methylene function, and stabilize the approaching anionic nucleophile. a-Haloacids and amides will exhibit a similar effect, and both iodoacetic acid and iodoacetamide have found use as biochemical probes for the alkylation of purified enzymes. 

Group-specific reagents there are a variety of reagents which can specifically modify functional groups found in enzymes, e.g. iodoacetate at pH 5.5 was employed to identify specific histidine residues important in the catalytic activity of pancreatic ribonuclease. The other histidine residues in this enzyme molecule are less reactive with iodoacetate. 

Price et al. (86). The evidence is based on experiments in which DNase I was reacted with iodoacetate at pH 7.2 in the presence of 0.1 M Mn2+. Under these conditions the enzyme is gradually inactivated and the loss of activity parallels the formation of one residue of 3-carboxymethyl histidine per molecule. The rate of the alkylation reaction is dependent on Mn2+ concentration. Substitution of Mn2 by Cu2+ in the presence of tris buffer greatly increases the rate of alkylation. A 29-residue peptide con-... 

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. 

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. 

Nitrogens 1 and 3 in the imidazole ring of histidyl residues in proteins may be alkylated with iodoacetic acid (generally in a much slower reaction than alkylation of cysteinyl residues) to give three carboxy-methyl derivatives 1-carboxymethylhistidine, 3-carboxymethyl-histidine and 1,3-dicarboxymethylhistidine ( 3.4). In general, the 3-carboxymethyl derivative is formed most rapidly. These derivatives are stable to acid hydrolysis under the usual conditions (but excess reagent must be removed) and may be analyzed on the long column of most analyzers as described below. 

Reagents capable of modifying the histidyl residue with complete specificity are not available to date. Reaction with a-haloacids and amides at near neutral pH offers the best approach to the modification of histidine in native proteins. In a protein such as insulin, which contains neither methionyl nor cysteinyl residues, reaction with iodoacetate at pH 5.6 leads to the formation of a derivative in which the sole modification is the N-carboxymethylation of two histidyl residues (Covelli et al. 1973). 

The imidazole ring of histidine also undergoes alkylation on reaction with iodoacetate. In ribonuclease, two residues (His 12 and His 119) are alkylated with loss of activity when the enzyme is treated with iodoacetate at pH 5.5. 

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