Serine Diisopropylfluorophosphate

Diisopropylfluorophosphate (DIFP) reacts with active-site serine ... 

Plasmin is a serine proteinase (inhibited by diisopropylfluorophosphate, phenylmethyl sulphonyl fluoride and trypsin inhibitor) with a high specificity for peptide bonds to which lysine or arginine supplies the carboxyl group. Its molecular weight is about 81 Da and its structure contains five intramolecular disulphide-linked loops (kringles) which are essential for its activity. 

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). 

Diisopropylfluorophosphate, in contrast to its action on other serine hydrolases, has only a slight inhibitory effect on alkaline phosphatase in the range 1-10 ml (76, 113). 

Diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), both organophosphorus inhibitors, are potent irreversible inhibitors of serine proteases. However, because of their additional inhibition of acetylcholinesterase these compounds are highly toxic [26]. Another toxic but potent trypsin inhibitor is (4-aminophenyl)-methanesulfonyl... 

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. 

Mechanism of action Isoflurophate [eye soe FLURE oh fate] (diisopropylfluorophosphate, DFP) is an organophosphate that covalently binds to a serine-OH at the active site of acetylcholinesterase (Figure 4.9). Once this occurs, the enzyme is permanently inactivated, and restoration of acetylcholinesterase activity requires the synthesis of new enzyme molecules. Following covalent modification of acetylcholinesterase, the phosphorylated enzyme slowly releases one of its isopropyl groups (Figure 4.9). The loss of an alkyl group, which is called... 

Diisopropylfluorophosphate] (organophosp hate) Synthetic insecticide AChE (forms phosphoryl ester with active site Serine)... 

Serine residues occurring in the active sites of serine proteases (e.g., chymotryp-sin) or other hydrolyases (e.g., acetylcholinesterase) are specifically modified by diisopropylfluorophosphate in a reaction that is essentially irreversible. 

Tyrosine Tryptophan Serine Aspartic Acid Glutamic Acid T etranitromethane Chloramine T iV-Bromosuccinimide 2-Hydroxy-5-nitrobenzyl bromide Diisopropylfluorophosphate Halomethyl ketones Carbodiimides Trimethyl oxonium fluoroborate Isoxazolium salts Chloramine T also modifies histidines and methionines... 

CMV protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Serl32 as the active site serine (4,5). The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132... 

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 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 absolute number of essential residues, either of the same or different types of amino acids, can frequently be determined. One of the simplest and best methods is the use of one of the several types of active-site labeling reagents which have an easily monitored group attached. Such a group could be labeled with a radioactive element or with a distinct optical absorption band. One that has frequently been used with the serine -type enzymes is diisopropylfluorophosphate labeled with 32P. 

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... 

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. 

The active center of A. has two parts, the anionic binding site for the quatemaiy nitrogen (which is responsible for the alcohol specificity) and the esterase center (where a catalytic serine and histidine lyse the ester bond). The enzyme is inactivated by blockage of either the serine hydroxyl (by organic phosphate esters, such as diisopropylfluorophosphate or diethyl p-nitrophenylphosphate), or the anionic center by tri-methylammonium derivatives. If the enzyme has been blocked by organophosphates, it can be reactivated by pralidoxime salti which are therefore used as antidotes to organophosphate poisoning. 

Amino acid residues of the A.c. are identified by specific labeling with coenzyme, or by reaction with inhibitors or reagents specific for particular side chains. Some widely used irreversible inhibitors of the catalytic center of serine proteases are tosyllysine chloromethyl ketone (TLCK), which reacts with histidine, and diisopropylfluorophosphate (DFF) and phenylmethane sulfonyl fluoride (PMSF), which forms esters with serine residues. 

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