34 1. lODOACETATE AND lODOACET AMIDE 



dine residues in ribonuclease is carboxymethylated and the rate is much 

 greater than for free histidine, indicating that this single histidine must be 

 in a particularly reactive state. The reactive histidine is the one nearest 

 the C-terminal end of the protein chain (position 119) (Stein and Barnard, 

 1959). Two chromatographically distinct monocarboxy methylated deriva- 

 tives are found when ribonuclease A is treated with iodoacetate at pH 5.5 

 (Crestfield et al., 1963). The major derivative is 1-carboxymethylhistidine- 

 119-ribonuclease, i.e., with the histidine in position 119 carboxymethylated 

 at the 1 -position of the imidazole ring, and the minor product is 3-carboxy- 

 methylhistidine-l'i-ribonuclease. Thus histidine residues in two different 

 regions of the enzyme are attacked at different sites on the imidazole ring. 

 The major product is inactive while the minor one is active. The variation 

 of the alkylation rate with pH has been treated satisfactorily by Lamden 

 et al. (1962) on the simple assumption that iodoacetate reacts with only 

 the singly ionized form of the enzyme, i.e., with EH in the equilibrium 

 EH2 ^ EH ±5 E. The piiCa's for the enzyme groups were calculated to be 

 4.85 and 5.55, which are quite low for imidazole, so they raise some doubt 

 whether the inhibition can be immediately related to reaction with the 

 histidine residues. Various treatments of ribonuclease — oxidation by per- 

 formate, denaturation with urea or guanidine — abolish the reactivity of 

 histidine with iodoacetate (Stark et al., 1961). Furthermore, iodoacetamide 

 does not react with this histidine at pH 5.5 and 40°, a marked difference 

 from iodoacetate; since both react with a-iV-acetyl-L-histidine at the same 

 rate, the difference in the enzyme must be due to the environment of the 

 histidine residue. The three-dimensional structure of the enzyme is neces- 

 sary for the activation of the histidine residue, and a positive charge was 

 postulated to be located nearby. Ribonuclease-S (subtilisin-modified ribo- 

 nuclease) consists of a protein portion and a 20-residue peptide. Inactivation 

 of the protein portion by iodoacetate at pH 6 is caused by the carboxy- 

 methylation of methionine residues, resulting in the inability to bind the 

 peptide and form an active enzyme (Vithayathil and Richards, 1961). No 

 reaction with histidine residues occurs under these conditions. However, 

 the total enzyme at pH 6 is reacted at a histidine residue on the protein 

 portion so that the peptide component is necessary for the reactivity of this 

 residue. One is thus led to conclude from these recent studies that iodoace- 

 tate can react with amino acid residues other than cysteine, although the 

 rates are slow unless very high concentrations of iodoacetate are used, but, 

 more important, that the reactivity of a particular amino acid depends 

 strongly on the steric and electrostatic factors arising from the environ- 

 ment on the enzyme surface. These results have introduced new methods 

 for the use of iodoacetate in studying enzyme mechanisms. 



