Iodoacetamide, enzyme inhibition

Assayed by coupling with the next reaction and following NADPH oxidation spectrophotometrically. SH-enzyme inhibited by iodoacetamide, etc. Protected by preincubation with acetyl-ACP not malonyl-ACP... 

The L-aminopeptidase is sensitive to various classes of proteinase inhibitors. Strong inhibition of the enzyme is observed by treatment with the thiol reagents / -chloromercu-ribenzoate (pCMB) and iodoacetamide. The inhibition by pCMB can be reversed by subsequent treatment with dithiothreitol. In addition, the enzyme is inhibited by the metalchelating compounds EDTA and o-phenanthroline and the serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. These phenomena point to an essential serine or cysteine residue in the active site furthermore, divalent cations seem to be involved in the catalytic mechanism and/or are important for the stability of the enzyme. 

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. 

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). 

Iodoacetamide, an a-halogenocarbonyl type of alkylating agent, will readily react covalently with -SH sulfhydryl groups. An enzyme s reactive site containing a cysteine residue may therefore be irreversibly inhibited if sufficient concentration can be achieved. 

Iodoacetamide itself does not inhibit this enzyme. However, when bonded to the salicylic acid molecule, which has some structural resemblance to lactic acid, irreversible alkylation and irreversible inhibition do take place. Since salicylic acid itself is a reversible inhibitor... 

The yeast enzyme has 8 86,378,379) to 9 368,399) free SH groups per subunit and is inhibited by thiol reagents. It may also be inactivated by oxidation to form intrachain disulfide bridges 399). Preparations may vary in SH content 366,399,408,. 09), although the number of cysteine residues reactive to iodoacetamide is constant 408,409). This type of variability seems reminiscent to that of the subfractions of rat liver alcohol dehydrogenase (Section II,A,3,a). 

Various protease inhibitors were incubated with the enzyme to determine what class of protease this enzyme might be. The serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), the thiol protease inhibitor iodoacetamide (lAA) and the metalloprotease inhibitor EGTA were incubated with the enzyme and compared to a control with no reagent. Results shown in Table 3 indicated that the enzyme is neither a thiol nor metalloprotease. However, the enzyme was inhibited significantly by PMSF, strongly suggesting that it is similar to a serine protease. 

More recently, Young and Conn (25) have studied the inhibitors of wheat-germ glutathione reductase. Almost complete inhibition of the enzyme was obtained with 10 M iodoacetic acid, KP M iodoacetamide, and 10 M p-chlormercuribenzoic acid. Partial inhibition was observed in each case when the inhibitors were tested at one-tenth the concentrations given. Prior incubation of the enzyme with GSSG did not protect the protein from inhibition, and prior incubation of the enzyme with the inhibitor did not increase the inhibition. 

The steps required to convert mevalonic acid to the active-isoprenoid intermediate have been worked out with some assurance. The initial step involves the phosphorylation of mevalonic acid to mevalonic acid-5-phosphate by an enzyme called mevalonic kinase. This enzyme was found in yeast by Tchen (1958). The properties of the mevalonic kinase of liver have been described in detail by Levy and PopjAK (1960). The kinase is inhibited by p-chloromercuribenzoate but not by iodoacetamide. The enzyme requires Mg++, Mn++, or Ca++ and ATP or inosine triphosphate. The kinase is specific for the (+) form of mevalonic acid. Mevalonic acid-5-phosphate is phosphorylated further to give mevalonic acid-5-pyrophos-phate (de Waard and Popjak, 1959 Henning et al. 1959). The purified enzyme (Bloch et al., 1959) requires a divalent metal ion for activity (Mg++ is preferable) and has no pronounced pH optimum. Mevalonic acid pyrophosphate then undergoes simultaneous dehydration and decarboxylation to yield isopentenylpyro-phosphate (Lynen et al., 1958 Chaykin et al., 1958). The enzyme concerned with the dehydration and decarboxylation has been purified (Bloch et al., 1959) and shown to have a pH optimum between 5.5 and 7.4 and to require a divalent metal ion (Mg++, Mn++, Fe++ or Co++). The series of reactions in which mevalonate is converted to isopentenylpyrophosphate is outlined in Figure 6. Brodie et al. (1963) have established a new pathway for the biosynthesis of mevalonic acid from malonyl CoA. The importance of this particular pathway in the synthesis of sterols is still unknown. 

Fig. 3. Protection of thiolase against iodoacetamide inhibition by preincubation with acetoacetyl-CoA or acetyl-CoA. >, Thiolase was incubated with 9 x 10" M acetoacetyl-CoA (AcAcCoA) or acetyl-CoA (AcCoA) for 10 minutes at 0°C and pH 8.2. 5 x 10" M iodoacetamide was then added and samples taken from the reaction mixture at the times indicated in the figure. The reaction of iodoacetamide with the enzyme was stopped by the addition of an excess of cysteine and...

The sulfhydryl nature of the mammalian glutaminase appears to be well established (853, 854). The following reagents inhibit mercuric chloride, p-chloromercuribenzoate, iodoacetamide, JV-ethylmaleimide, p-benzoquinone, and quinone. p-Chloromercuribenzoate inhibition is independent of phosphate concentration and appears to be a competitive inhibitor of glutamine, indicating that the —SH group of the enzyme is a site of attachment for the substrate (854)-... 

One of the last steps in flavonoid synthesis is methylation and an 0-methyl-transferase for this has been purified 82-fold from cell suspiension cultures of parsley. It has a pH optimum of 9.7, requires Mg, and the molecular weight is ca. 48000. Unlike catechol methylase from animal tissues, it is not inhibited by p-chloromercuribenzoate or by iodoacetamide. Luteolin (27) and its 7-glucoside were the best substrates, giving chrysoeriol (28) and its 7-gluco-side respectively K values were 4.6 x 10" and 3.1 x 10 moll Erio-dictyol and caffeic acid were poor substrates (K values 1.2 x 10 and 1.6 X 10 moll , respectively) and the enzyme was quite spteciflc for catechols and only 0-methylated a m-hydroxy-group. 

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