Iodoacetic enzyme inhibitor

Hayes compared the toxicity of the chloroacetate, fluroacetate, and iodoac-etate in rats. The 24-h LD50 values were 108, 5, and 60 mg/kg, respectively. LD90 doses were delivered to rats and the time until death (LT) was determined. The LT50 for chloroacetate, fluroacetate, and iodoacetate was 130,310, and 480 min, respectively. Based upon this comparative study, fluroacetate was the most toxic, iodoacetate the intermediate, and chloroacetic acid was the least toxic of the three compounds. Bromoacetic acid is not as well studied although it is a potent enzyme inhibitor. 

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. 

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],... 

The insensitivity of the type II enzyme to PTU seems to exclude the generation of an enzyme SI intermediate as is the case with the type I deiodinase (see Section 2.4). The lack of involvement of a catalytic enzyme SH group in type II deiodination is also suggested by the weak effects of iodoacetate [82], a potent inhibitor of the type I deiodinase. It may be speculated that the type II enzyme catalyses the transfer of I+ from the substrate directly to the SH group of the cofactor [82], In contrast to PTU, iopanoic acid has similar inhibitory effects on the type I and II deiodinases [71-73,84,89]. 

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Von Euler and Adler (1935) reported that 5 X 10 M monobromoacetate inhibited 50%. IX 10 M iodoacetate reduced the activity by 92% the degree of inhibition was a function of time of contact between inhibitor and enzyme (Dixon, 1937). Lutwak-Mann (1938) found that the yeast ADH was inhibited by cyanide, whereas the mammalian and bacterial enzymes were not. Urethane, oxalate, maleate, and pyrophosphate similarly inhibit yeast ADH quinine, morphine, and nicotine did not. Von Euler and Adler (1935) found no effect with sodium fluoride. 

IRREVERSIBLE INHIBITION Inhibition may be reversible or irreversible. In reversible inhibition (i.e., competitive, uncompetitive, and noncompetitive inhibition), the inhibitor can dissociate from the enzyme because it binds through noncovalent bonds. Irreversible inhibitors usually bond covalently to the enzyme, often to a side chain group in the active site. For example, enzymes containing free sulfhydryl groups can react with alkylating agents such as iodoacetate ... 

Only a few enzymes are specifically secreted by organs into the blood stream. These are the enzymes of blood coagulation, cholinesterase, and amylase [21]. Serum, in the main, is a passive receptacle for enzymes derived from the tissue cells and the formed elements of blood. Normally, the level of enzymes in serum is both low and constant. Since most of the enzymes present are derived from cells, it follows that these enzymes must be able to pass through the limiting membrane of the parent cell. This outward passage is accomplished either by diffusion through pores or alternatively by the aid of an active transport system. Except in those cases where enzyme secretion is a physiological process, active transport is unlikely. The loss of enzymes from cells is accelerated by tissue injury, and also by metabolic inhibitors like 2,4-dinitrophenol, iodoacetate, and carbon monoxide [22]. The implication that the retention by the cell of a... 

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. 

Several compounds are potent inhibitors of tyrosine oxidation probably the most significant of these is diethyldithiocarbamate. This is considered primarily as a copper binder and suggests that copper has a role in one or more of the reaction-steps of tyrosine oxidation. This is further supported by the fact that the copper counteracts the inhibition. Other strong inhibitors were iodoacetate, which suggests that sulfhydryl groups are essential for at least one of the enzymes, and hydroxylamine. The action of the latter suggests either a metal ion function or the binding of an enolic intermediate. ... 

SaroHnycin (Fig. 1), a streptomycete antibiotic inhibits the DNA polymerase. It prevents the incorporation of thymidine into intact tumour cells, and into DNA primer in cell-free extracts. Sarcomycin probably blocks the thiol groups which are indispensable for the activity of the DNA-polymerase molecule. It is a noncompetitive inhibitor of the enzyme in the same way as iodoacetate or iodo-acetamide. Its action is prevented or reversed by 2-mercaptoethanoL... 

Sephadex G-75 and SDS polyacrylamide gel electrophoresis. There was no evidence for a subunit structure of the three similar enzymes due to SDS polyacrylamide gel electrophoresis after treatment with 1% 2-mercapto-ethanol and 1% SDS in 250 mM phosphate buffer, pH 7.1, at 90°C for 15 min. The apparent Km-values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect (Table 2). Competitive product inhibition was found for AMP, with Ki-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was pi,P -di(adenosine-5 )pentaphosphate (=DAPP) with K -values of 0.020 jjM for R. palustris and R. sphaeroides, and 0.15 jjM for R. rubrum. Sulfhydryl reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. Several divalent cations were effective in potentiating the enzymatic activity. The order of decreasing effectiveness was Mg2 > Ca > Co > Mn " " > Zn " ". The adenylate kinases from all three bacteria studied were activated maximally by a molar ratio of Mg2+ ADP of 1.0. 

Of a series of conventional inhibitors tested with the bacterial enzyme, only p-chloromercuribenzoic acid caused significant loss of activity, and this was prevented by glutathione. The Neurospora enzyme was not affected by iodoacetate, A -phenylmaleimide, semicarbazide, or cyanide (Ottey and Tatum, 1956). It is noteworthy that neither inhibition nor activation studies gave any evidence for a metal cofactor in the Pseudomonas preparation. An enzyme from Nocardia that is also inhibited by organic mercury also showed no evidence for a metal component (Cain and Cartwright, 1960). An iron requirement has been found for the Neurospora enzyme, however. When sufficiently pure preparations are available, it will be of interest to analyze the other protocatechuic oxidases for bound metal. 

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