Diisopropylfluorophosphate enzyme inhibitor

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. 

The importance of these amino acid side chains is illustrated by the action of two kinds of irreversible enzyme inhibitors (shown in Figure 7-9). Diisopropylfluorophosphate transfers its phosphate to the active site serine. The resulting phospho-enzyme is totally inactive. Chloromethyl ketones alkylate the active site histidine. 

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. 

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],... 

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. 

In the absence of any specific information about the nature of such activities, it is often best to mix a cocktail containing several or all of the available proteolytic inhibitors. These include such compounds as 1,10-phenanthroline, soybean trypsin inhibitor, leupeptin, benzamidine, antipain, aprotinin, phenyl-methanesulfanyl fluoride, and diisopropylfluorophosphate. As a precaution against any enzyme destruction, such a cocktail is best added to the buffer in which the cellular lysis will be carried out (Fig. 5.6). 

The most thoroughly investigated pancreatic lipase is from the pig. Most of our knowledge of the structure and properties of this enzyme comes from P. Desnuelle and his co-workers in Marseille (3) (Table III). The lipase occurs in two similar forms, isoenzymes Lipase A and Lipase B. There is no known proenzyme (zymogen). The enzyme does not react with diisopropylfluorophosphate (DFP), the standard inhibitor... 

The compounds diisopropylfluorophosphate (DIFP) is a potent inhibitor of the proteolytic enzyme ch)motrypsin. It permanently inactivates the... 

Diisopropylfluorophosphate, which irreversibly binds to active serine residues on some hydrolytic enzymes, is an example of this type of inhibitor, decreases because some enzyme is completely removed from the system. (Remember, = j>rE]i.) An irreversible inhibitor can be dis-... 

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]. 

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. 

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. 

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. 

---