Iodoacetates, enzyme

Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in the glycolytic pathway (Chapter 8), is inactivated by alkylation with iodoacetate. Enzymes that use sulfhydryl groups to form covalent bonds with metal cofactors are often irreversibly inhibited by heavy metals (e.g., mercury and lead). The anemia in lead poisoning is caused in part because of lead binding to a sulfhydryl group of fer-rochelatase. Ferrochelatase catalyzes the insertion of Fe2+ into heme. 

Figure 17-3. Mechanism of oxidation of giyceraldehyde 3-phosphate. (Enz, glycer-aldehyde-3-phosphate dehydrogenase.) The enzyme is inhibited by the— 5H poison iodoacetate, which is thus abie to inhibit glycolysis. The NADH produced on the enzyme is not as firmly bound to the enzyme as is NAD. Consequently, NADH is easily displaced by another molecule of NAD". ...

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Identification of the energy source for muscle contraction and determination of the order in which the phosphate esters were metabolized was helped by the use of inhibitors. These inhibitors blocked different stages in glycolysis and caused preceding substrates to accumulate in quantities which could greatly exceed those normally present. The compounds were then isolated, identified, and used as specific substrates to identify the enzymes involved in their metabolism. Iodoacetic acid (IAc) was one of the most important inhibitors used to analyze glycolysis. 

The basis of the action of iodoacetate on muscle contraction was uncovered by Dickens and Rapkine (ca. 1933). They found iodoacetate alkylated SH groups on proteins, especially those in glyceraldehyde 3-phosphate dehydrogenase (G-3-PDH). When the enzyme was inhibited precursors accumulated—hexose mono- and diphosphates—as in Lundsgaard s experiments. 

Analyses of enzyme reaction rates continued to support the formulations of Henri and Michaelis-Menten and the idea of an enzyme-substrate complex, although the kinetics would still be consistent with adsorption catalysis. Direct evidence for the participation of the enzyme in the catalyzed reaction came from a number of approaches. From the 1930s analysis of the mode of inhibition of thiol enzymes—especially glyceraldehyde-phosphate dehydrogenase—by iodoacetate and heavy metals established that cysteinyl groups within the enzyme were essential for its catalytic function. The mechanism by which the SH group participated in the reaction was finally shown when sufficient quantities of purified G-3-PDH became available (Chapter 4). 

The surest way to inhibit an enzyme is to block the active site irreversibly by chemical reaction with some active species to form a covalent bond. Thus, iodoacetate will irreversibly inactivate thiol proteases by forming the stable carboxymethyl mercaptan. lodoacetate is of course non-selective (many other enzymes would be inactivated), toxic (many sensitive sites would be alkylated) and moreover the drug itself is unstable due to its very reactivity. 

Predict which Calvin cycle enzyme(s) would be inhibited by iodoacetate, and explain why. 

Carboxymethylation. It was found by Vallee and Li that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate.1405 Since this reaction causes deactivation of the enzyme, this cysteine residue, later identified as Cys-46,1406 was suggested to be at the active site. The deactivated carboxymethylated enzyme still binds NAD+. The carboxymethylation of this residue is preceded by a reversible binding of iodoacetate to the enzyme.1407 This observation has helped to identify an anion-binding site in the coenzymebinding domain, where the pyrophosphate group of the coenzyme binds. 

Iodoacetate had no effect on the rabbit muscle enzyme (130, 135) but did inhibit the carp muscle and pea seed enzyme (48, 136). Organic mercurials are also reported to inhibit the enzyme from several sources (48, 125, 126, 130, 136). Except for the preliminary report by Wolfenden et al. (92) that mercurials desensitized the rabbit muscle enzyme to allosteric inhibition by GTP, the role of sulfhydryl residues in AMP aminohydrolase is not understood. 

Iodoacetic acid, A-bromosuccinimide, and H202 were found to be strongly inhibitory, whereas iodoacetamide was only slightly inhibitory and diisopropylfluorophosphate was not inhibitory. These results suggest that tryptophan, methionine, and/or histidine, but not serine, are involved in the enzymic activity (43). 

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-... 

Inhibition by a variety of metal-binding agents competitive with respect to phosphoryl substrates (118-120) has suggested that an enzyme-bound divalent cation (other than Mg2+) may participate also in the binding of phosphate substrates. Observed inhibition by p-chloro-mercuriphenyl sulfonate and iodoacetate suggests the possibility that sulfhydryl groups may also be involved at, or near, the active enzymic site (119, 120). 

Other sulfhydryl reagents, such as p-mercuribenzoate and iodoaceta-mide, produced similar activation (44), except that with these compounds increases in activity were also observed at pH 9.1 (Table I). With p-mercuribenzoate maximum activation was observed when 2-4 sulfhydryl groups were titrated, and with excess reagent catalytic activity was almost completely abolished (44)- Similar results were obtained with FDNB (15). The reactive sulfhydryl groups may be located in apolar regions of the enzyme molecule since they were not affected by N-ethylmaleimide or 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. 

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. 

When RNase A is treated with iodoacetate (ICH2CO2 ), the two major products obtained are carboxymethylated derivatives of His 12 and His 119. Both of these enzymes are severely inhibited, which suggests that both His 12 and His 119 are important in the active site. The enzyme also is completely inhibited by the reaction of Lys 41 with fluorodinitrobenzene. 

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... 

Competitive inhibition of the carboxypeptidase from A. saitoi by small substrates was found with hydrocinnamic acid, indole-3-propionic acid, and 4-phenylbutyric acid [80], The K for hydrocinnamic acid inhibition was 4 x 10 4 M. Diisopropylfluorophosphate (DFP) and tosyl-L-phenylalanylchloromethane (TPCK) were also powerful inhibitors of the carboxypeptidase from A. oryzae (80). />-Chloromercuribenzoate (PCMB) and iodoacetic acid were also powerful inhibitors of the carboxypeptidase from A. saitoi, while the inhibitors of DFP, TPCK, PCMB, and iodoacetic acid on the carboxypeptidase from A. saitoi were less than that of A. oryzae [80], As the carboxypeptidase activity of A. saitoi has no effect when used with ethylenediaminetetraacetate (EDTA) and o-phenanthroline, the enzyme is a different type of carboxypeptidase from those of the pancreatic sources, carboxypeptidase A and carboxypeptidase B [80],... 

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