Iodoacetate lysine residues

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. 

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... 

Both thee-N-monocarboxymethyl and the e-N-dicarboxymethyl derivatives of lysine are formed by reaction of proteins with iodoacetate at alkaline pH values at which lysyl residues are at least partially un-protonated ( 3.8.2). Both derivatives are stable to acid hydrolysis under the usual conditions. 

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