Iodoacetamide, enzyme

A failure by one of us to take fully into account the presence of inactivated xanthine oxidase, leading to misinterpretation of incomplete reaction of enzyme with iodoacetamide and hence to the apparently erroneous conclusion, that the two FAD molecules in the enzyme were non-equivalent (72), may serve as a warning to others. This reagent has since been shown to alkylate the flavin of reduced xanthine oxidase molecules, whether these are of the active or inactivated forms (73). Thus, under conditions where little of the inactivated form is reduced, the reagent becomes a specific one for the active enzyme (20). In the original experiments (59, 72) the content of active enzyme was, by coincidence, rather close to half of the total enzyme present. Thus, the presence of inactivated enzyme, rather than a lack of reactivity of one... 

Subsequently this mechanism, known as the affinity labelling mechanism, has also been shown to operate with 2-bromopropionate, 3-bromopropionate and 2-bromobutyrate in a similar way. Iodoacetamide also deactivates the enzyme, but by direct alkylation.1408... 

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). 

The activation of liver FDPase by a variety of sulfhydryl reagents has been examined by Little et al. (46), and their results generally confirm those reported by Pontremoli and his co-workers. In the disulfide exchange reaction 5,5 -dithio-bis(2-nitrobenzoic) acid was most effective (45), and in general, the disulfides were more effective than reagents such as p-mercuribenzoate or iodoacetamide. The nature of the group introduced appears to affect the conformation of the modified enzyme. 

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. 

Active-site directed inhibitors have reactivity with the enzyme greatly enhanced over that of non-specific inhibitors thus phenacyl iodide inhibits papain 50-fold faster than iodoacetamide whereas the active-site directed inhibitor 4-toluenesulphonylamidomethyl chloromethyl ketone reacts some 650-fold faster. The enhanced rate is due to complexation of the inhibitor with the enzyme, and indicates that the inhibitor must be reacting at the active site. 

Over a decade ago, work on the enzyme aldolase reductase elegantly demonstrated this point. The noncovalent inhibitor alrestatin was modified to contain various electrophiles a-chloroacetamide, a-bromoacetamide or a-iodoacetamide. Noncovalent interactions between inhibitors and protein would not have changed, but molecules behaved differently based on the electrophile the weakest showed reversible inhibition, whereas the iodoacetamide displayed almost complete irreversible inhibition.1401 These results are an important warning if a reaction is too facile, irreversible reactions can obscure true binding affinities. 

An irreversible inhibitor binds tightly, often covalently, to amino acid residues at the active site of the enzyme, permanently inactivating the enzyme. Examples of irreversible inhibitors are diisopropylfluorophosphate (DIPF), iodoacetamide and penicillin. 

Ser residue in the active site of the enzyme acetylcholinesterase, irreversibly inhibiting the enzyme and preventing the transmission of nerve impulses (Fig. la). Iodoacetamide modifies Cys residues and hence may be used as a diagnostic tool in determining whether one or more Cys residues are required for enzyme activity (Fig. lb). The antibiotic penicillin irreversibly inhibits the glycopeptide transpeptidase enzyme that forms the cross-links in the bacterial cell wall by covalently attaching to a Ser residue in the active site of the enzyme (see Topic Al). 

The type I deiodinase of liver and kidney is inactivated by different SH-selective reagents. In particular, it shows an extremely high susceptibility to carboxymethy-lation by iodoacetate and a somewhat lesser sensitivity for iodoacetamide and bro-moacetate [42]. In comparison, A-alkylmaleimides are only inhibitory at high concentrations (> 0.1 mM) [42]. Enzyme inactivation by iodoacetate follows pseudo... 

There remains model 4, and MacQuarrie and Bernhard 175) have utilized the full-site reactivity by iodoacetamide and half-site reactivity by FAP to provide support for this model. Thus, di(2-furylacryloyl) enzyme was prepared, and the two remaining sites were blocked with iodoacetate. Acyl groups were then removed from this derivative by arsenolysis, and the resulting dialkyl enzyme was tested for stoichiometry with FAP. Only one acyl group could be incorporated into the dialkyl enzyme. This result cannot be explained in terms of an induced asymmetry model, and indeed, can only be explained by a preexisting asymmetry model if there is a subunit rearrangement. In addition, alkylation of the enzyme with varying quantities of iodoacetate, followed by acylation of these derivatives with FAP, showed a 2 1 ratio of alkylation to acylation, independent... 

Elegant experiments, which capitalized on the ability of iodoacetamide to specifically alkylate the active site cysteine of the -ketoacyl synthase, were performed, which definitively proved the capability of the yeast FAS in decar-boxylating malonyl CoA [75]. Following alkylation, FAS activity is abolished however, the enzyme still transacylates malonyl CoA to the phosphopantetheine thiol, where it is decarboxylated before being transferred back to CoA by the transacylase prior to its release as acetyl CoA. 

MSAS from P. patulum was separated from the FAS via sucrose gradient centrifugation [121,122] and thus shown to constitute a distinct multifunctional enzymatic system. It was purified to homogeneity and found to be a 190 kDa multifunctional enzyme [22,120]. The enzyme was more stable in the presence of its substrates and at mildly basic pH values. The pH optimum of the enzyme was 7.6 and apparent K values for its substrates were 10 pM (acetyl-CoA), 7 pM (malonyl CoA), and 12 pM (NADPH) [115,120,123]. The rate for triacetate lactone formation in the absence of NADPH was determined to be ten-fold lower than for 6-MSA formation (Fig. 5) [120]. Analogous to FASs and peptide synthetases, 4 -phosphopantetheine is a covalently bound cofactor of 6-MSAS [124]. Likewise, iodoacetamide and N-ethylmaleimide were found to inactivate the enzyme, suggesting the presence of catalytic sulfhydryl residues in 6-MSAS [124]. Furthermore, in the presence of malonyl CoA and NADPH, low concentrations of iodoacetamide convert 6-MSAS into a malonyl CoA decarboxylase. Without external addition of acetyl-CoA, 6-MSAS decarboxylates the malonyl group and the derived acetyl moiety is used as a starter unit for the formation of 6-MSA [125]. 

LO was purified from two weeks-old bovine calf aorta by a modification of published procedures (14, 15). The aortas (ca. 600 g) were cleaned and finely ground prior to protein extraction. Extractions and all subsequent procedures were carried out at 4 °C. The ground aortas were extracted with 0.15 M NaCl-16 mM potassium phosphate (KPi) (saline buffer), 16 mM KPi, and 4 M urea-16 mM KPi buffer (pH 7.8), sequentially, in the presence of protease inhibitors PMSF (1 mM) and iodoacetamide (0.04% w/v). The saline buffer and KPi extracts contained negligible activity and were discarded. The urea buffer extracts were pooled (total of 4 to 5 L) and mixed with hydroxyapatite gel (100 g, preequilibrated in 4 M urea-16 mM KPi). After batch elution from hydroxyapatite, the urea-soluble enzyme solution was concentrated (ca. 700 ml) and dialyzed against 16 mM KPi (pH 7.8) buffer. 

Exactly 1.7 mg of a purified enzyme (MW =55,000) was incubated with an excess of iodoacetamide-C (S.A. = 2 fiCii mmole). The car boxy methylated protein was then precipitated, washed free of unreacted iodoacetamide-C , dissolved in a small amount of buffer, and the entire solution counted in a scintillation counter operating at 80% efficiency. In one hour, the sample gave 13,190 counts above background. Calculate the number of reactive SH groups per molecule of protein. 

The enzyme preparation from Aerobacier aerogenes was found by Hogness and Battley to be inactivated by oxygen, iodoacetamide, p-(chloromercuri)-benzoate, and A -ethylmaleimide. After inactivation with oxygen, activity could be recovered on treatment with mercaptoacetate, cysteine, sodium sulfide, sodium cyanide, or 2-mercaptoethanol." The authors, however, have not drawn any conclusion as to whether these observations should be taken to indicate that sulfhydryl groups are involved in the enzyme activity. 

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