Diisopropylfluorophosphate, enzyme inhibition

The previous discussion of amino acid catabolic disorders indicates that catabolic processes are just as important for the proper functioning of cells and organisms as are anabolic processes. This is no less true for molecules that act as neurotransmitters. To maintain precise information transfer, neurotransmitters are usually quickly degraded or removed from the synaptic cleft. An extreme example of enzyme inhibition illustrates the importance of neurotransmitter degradation. Recall that acetylcholine is the neurotransmitter that initiates muscle contraction. Shortly afterwards, the action of acetylcholine is terminated by the enzyme acetylcholinesterase. (Acetylcholine must be destroyed rapidly so that muscle can relax before the next contraction.) Acetylcholinesterase is a serine esterase that hydrolyzes acetylcholine to acetate and choline. Serine esterases have catalytic mechanisms similar to those of the serine proteases (Section 6.4). Both types of enzymes are irreversibly inhibited by DFP (diisopropylfluorophosphate). Exposure to DFP causes muscle paralysis because acetylcholinesterase is irreversibly inhibited. With each nerve impulse, more acetylcholine molecules enter the neuromuscular synaptic cleft. The accumulating acetylcholine molecules repetitively bind to acetylcholine receptors. The overstimulated muscle cells soon become paralyzed (nonfunctional). Affected individuals suffocate because of paralyzed respiratory muscles. 

The enzyme responsible for the biotransformation of capecitabine to 5 -deoxy-5-fluorocytidine (a precursor to 5-fluorouracil) was evaluated using purified enzyme, cytosol, and microsomes. The purified CES cytosolic enzyme, inhibited by the carboxylesterase inhibitors bis-nitrophenyphosphate and diisopropylfluorophosphate, was identified as belonging to the subgroup CES lAl based on the result of the N-terminal amino acid sequence. 

FIGURE 6-16 Irreversible inhibition. Reaction of chymotrypsin with diisopropylfluorophosphate (DIFP) irreversibly inhibits the enzyme. This has led to the conclusion that Ser195 is the key active-site Ser residue in chymotrypsin. 

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],... 

Diisopropylfluorophosphate (DFP) is an artifically produced (nerve gas-like) compound that inhibits serine proteases and esterases. Inactive enzymes may be activated by dissociation into subunits such as is the case with cAMP-dependent protein kinases. 

The activation of BSSL is specific to primary bile salts. Bile salts, secondary as well as primary, protect BSSL against inactivation by intestinal proteinases. BSSL is inactivated by heating at 50°C for 1 h, but sodium taurocholate prevents loss of activity. The enzyme is stable in buffer for 1 h at 37°C between pH 3.5 and 9. It is inhibited by blood serum, 1M NaCl, protamine sulphate, eserine and diisopropylfluorophosphate (Hernell, 1975 Blackberg and Hernell, 1983). 

As with the other enzymes in the GPI anchor biosynthetic pathway, little is known concerning the phosphoethanolamine transferase. However, the gene responsible for the defect in phosphoethanolamine transfer in the Class F Thy-1 mutants has been cloned [89]. The 917 base pair sequence encodes for a predicted hydrophobic protein of 219 amino acids. Phosphoethanolamine transferase activity in trypanosome cell lysates is inhibited by active site serine-directed inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate [90]. 

Enzyme inhibitors are of various types. For example, we can distinguish between irreversible and reversible inhibition. Sometimes an inhibitor reacts so strongly with the active center of an enzyme that the process cannot easily be reversed. Diisopropylfluorophosphate (DFP), for example, undergoes an irreversible reaction with the active centers of certain enzymes, with the liberation of hydrogen fluoride. One of the enzymes with which it reacts is cholinesterase, which is responsible for the functioning of the nerves. As a result DFP is a very powerful nerve gas. Poisons like potassium cyanide exert their action by the irreversible inhibition of enzymes which catalyze oxidative reactions. 

Covalent inhibition involves the chemical modification of the enzyme so that it is no longer active. For example, the compound diisopropylfluorophosphate reacts with many enzymes by adding a phosphate group to an essential serine hydroxyl group in the enzymes active sites. When phosphorylated, the enzyme is totally inactive. Many useful pharmaceutical compounds work by covalent... 

Enzyme inhibitors are often poisonous. For example, diisopropyl-fluorophosphate is a nerve poison because the enzyme acetylcholinesterase has a reactive site serine. Chymotrypsin and acetylcholinesterase are both members of the class of enzymes known as serine esterases, which are all inhibited by diisopropylfluorophosphate. 

Except for the inhibitory effects of thiocyanate and other anions, not many specific inhibitors have been reported. The enzyme from gastric mucosa is inhibited by OH-group-directed inhibitors, such as diisopropylfluorophosphate and methane-sulfonyl chloride and by the modifying solvent DjO [6]. Sulfhydryl reagents, like p-chloromercuribenzoate [4,20] and HgCl2 [5], inhibit the anion-sensitive ATPase from gastric mucosa and pancreas. 

Considerable advance has recently been made in the purification of bovine thrombin with the demonstration by Rasmussen (1955) that most of the active component of commercial thrombin preparations can be separated from the inactive protein impurities by chromatography on columns of Amberlite IRC-50 resin. This enzyme can be inhibited with diisopropylfluorophosphate (DFP) (Gladner and Laki, 1956 Miller and Van Vunakis, 1956) and reactivated by nucleophilic reagents such as hydroxamic acid (Gladner and Laki, 1956). On the assumption that DFP and thrombin combine in a mole ratio of 1 1 a molecular weight of 13,700 can be calculated (Gladner el al., 1958). The DFP is bound to a seryl residue, the sequence around which bears remarkable similarities to those of other enzymes which combine with DFP, as shown in Table XIII. 

Organophosphates - As with the carbamates, studies of the mechanism of action and elimination have been performed. Kinetic studies indicate that dealkylation in a series of organophosphates is a first order rate.95 The inhibitory action of diisopropylfluorophosphate (DFP) was found to be related to its affinity toward the enzyme both inhibition and affinity greater for serum cholinesterase than for erythrocyte acetylcholinesterase.9 The effect of substituent groups on phosphate esterase has been kinetlcally determined and electronic, steric and hydrophobic factors are implicated in their action.97 studies indicate that pH levels above 8.5 decrease inhibitory actions of phosphate esters and carbamates.98... 

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. 

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