Diisopropyl fluorophosphate and

This enzyme [EC 3.4.16.5] (also known as serine-type carboxypeptidase I, cathepsin A, carboxypeptidase Y, and lysosomal protective protein) is a member of the peptidase family SIO and catalyzes the hydrolysis of the peptide bond, with broad specificity, located at the C-terminus of a polypeptide. The pH optimum ranges from 4.5 to 6.0. The enzyme is irreversibly inhibited by diisopropyl fluorophosphate and is sensitive to thiolblocking reagents. 

Many compounds have been tested simultaneously with (2-chloroethyl)tri-methylammonium chloride on Thatcher wheat, to ascertain, if possible, whether these chemicals were affecting a particular metabolic process. Other cholinesterase inhibitors such as eserine, diisopropyl fluorophosphate, and nitrogen mustard, neither negated the effect from (2-chloroethyl)trimethylammonium chloride nor altered the growth of the plant themselves. Many other substances were also without effect on the action of (2-chloroethyl)trimethylammonium chloride. A very slight reversal of the alteration by (2-chloroethyl)trimethylam-monium chloride was obtained by 10 2 and 10 3 M choline, betaine, and adenine. Only gibberellin completely and rapidly reversed the shorter growth pattern of a plant which had been treated with (2-chloroethyl)trimethylammonium chloride. 

Inhibition Studies. A number of compounds were employed to study the amino acid residue(s) that are important for cellulase activity. Samples of enzyme (0.1 mL, 500 units) were pre-incubated with 0.1 mL of inhibitor in semimicroviscometers for 8 min at 35°C. CM-cellulose solution (0.8%, w/v), which had been separately equilibrated at 35°C for 20 min was added to the viscometers and initial viscosity losses were measured after 15 min. Inhibitors were replaced by buffer in control experiments. Compounds that are insoluble in buffer, e.g., N-ethylmalei-mide, diisopropyl fluorophosphate, and succinic anhydride, were dissolved in a small volume of 95% ethanol before assay. p-Chloromercuribenzoate (p-CMB) was first dissolved in 0.2M NaOH and the pH adjusted to eight prior to pre-incubation with cellulases. 

Henderson, J.D., R.J. Higgins, J.C. Dacre, et al. 1992. Neurotoxicity of acute and repeated treatments of tabun, paraoxon, diisopropyl fluorophosphate and isofenphos to the hen. Toxicology 72 117-129. 

Grigoryan, H., Schopfer, L.M., Thompson, C.M., Terry, A.V., Masson, P., Lockridge, O. (2008). Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin a potential mechanism of long term toxicity by organophos-phoms agents. Chem. Biol. Interact. April 22. (Epub ahead of print)... 

Fig. 2.1 shows the deduced amino acid sequences of human type I PLAj including PS-PLAi. The sequence homology of PS-lh,A, is about 80.0% between human [23] and rat [17]. The deduced amino acid sequence contains a catalytic triad composed of active Ser, Asp and His residues, and lid surface loops (Fig. 2.1). Interestingly, conventional lipases have long lid (22-23 residues) and / ) loops (18-19 residues), whereas PS-PLAi has a shorter lid (12 residues) and a shorter (deleted) fS9 loop (13 residues) [17, 23]. Both loops, which may be supported by S-S bonds of 14 cysteine residues, have been implicated in substrate recognition. These findings are compatible with the inhibition of the enzyme by diisopropyl-fluorophosphate and difhiothreitol [21].

V8. Volpe, M. T., Bisso, G. M., and Michalek, H., In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rats brain acetylcholinesterase. Neurochem. Res. 15, 975-970 (1990). 

Further non-specific inhibitors include the heavy metal ions Cu ", Fe ", and Hg " (Nees et al 1976) as well as p-chloromercuribenzoate, diisopropyl-fluorophosphate and o-phenanthroline (Schauer and Wember 1971). 

Inhibitors which interact only with peptidases of one catalytic type include pepstatin (aspartic peptidases) E64 (cysteine peptidases from clan CA) diisopropyl fluorophosphates (DFP) and phenylmethane sulfonyl-fluoride (PMSF) (serine peptidases). Bestatin is a useful inhibitor of aminopeptidases. 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

The mechanism by which A-esterases hydrolyze organophosphates is not completely understood. Involvement of a phosphorylated active-site cysteine and displacement of an activated H20 molecule are two possible hypotheses (see Sect. 3.7.1) [56], A-Esterases comprise enzymes that hydrolyze aryl esters, paraoxon (2.2) and related organophosphate pesticides, and diisopropyl-fluorophosphate (DFP, diisopropyl phosphorofluoridate, 2.3) and related compounds, including nerve gases. These enzymes are found in the current nomenclature listed under arylesterases, aryldialkylphosphatase, and diisop-ropyl-fluorophosphatase. 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

The organophosphorus cholinesterase inhibitors like diisopropyl fluorophosphate, phospholine, parathion, malathion etc. are highly toxic compound and cause irreversible inhibition of both true and pseudocholinesterases. They are highly lipid soluble compound and can easily cross the blood-brain barrier. 

Mazur and Bodansky (1946) found that diisopropyl fluorophosphate (DFP) irreversibly inhibits acetylcholine esterase. In particular, in 1949 Jansen, Balls, and their collaborators demonstrated the stoichiometric reaction of DFP with chymotrypsin (Jansen et al., 1949a,b Aldridge, 1950). 

Other chemicals which inhibit milk lipase include hydrogen peroxide, animal cephalin, sodium arsenite, diisopropyl fluorophosphate, 2,4 din-itro-l-fluorobenzene, p-hydroxymercuribenzoate, potassium dichromate, lauryl dimethyl benzyl ammonium chloride, aureomycin, penicillin, streptomycin, and terramycin (Schwartz 1974). 

The mammalian serine proteases have a common tertiary structure as well as a common function. The enzymes are so called because they have a uniquely reactive serine residue that reacts irreversibly with organophosphates such as diisopropyl fluorophosphate. The major pancreatic enzymes—trypsin, chymotrypsin, and elastase—are kinetically very similar, catalyzing the hydrolysis of peptides... 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

A wide variety of esters is hydrolyzed with the same Vmax (Table 7.8),37 constant product ratios are found (Table 7.9) stopped-flow studies using p-nitrophenyl phosphate find a burst of 1 mol of p-nitrophenolate ion released per enzyme subunit and the enzyme is covalently labeled by diisopropyl fluorophosphate.38... 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

The reversible inhibitors, which have a short to moderate duration of action, fall into two categories. Type one, exemplified by edrophonium, forms an ionic bond at the anionic site and a weak hydrogen bond at the esteratic site of acetylcholinesterase. Type two, exemplified by neostigmine, forms an ionic bond at the anionic site and a hydrolyzable covalent bond at the esteratic site. The irreversible inhibitors, exemplified by organophosphorus compounds (diisopropyl fluorophosphate, parathion,... 

The enzyme DGAT has not been purified to date, probably because it is a hydrophobic and integral membrane protein. Therefore, DGAT activity was measured using rat liver microsomes as an enzyme source and radiolabeled palmitate as a substrate by the method of Mayorek and Bar-Tana [52] with some modifications [53], The reaction mixture contains microsomal protein, BSA, [14C]palmi-toyl-CoA, MgCl2, diisopropyl fluorophosphate, 1,2-dioleoyl-vw-glyccrol, and a test sample in a total volume of 0.2 ml. After a 15-min incubation at 23°C, lipids are extracted and separated by thin-layer chromatography (TLC). The distribution of radioactivity on TLC is analyzed with a radioscanner to determine the amount of [14C]TG. 

The mixture was then cooled to 20°C and 19 gallons of benzene was added. This was followed by the introduction of 123.5 lb (2.80 Ib-mols) of dry powdered sodium fluoride (95% pure). The mixture was stirred and heated to the refluxing temperature in a period of 1 hour and held at this temperature (95° to 98°C) for 4 hours. The product obtained was cooled and filtered to yield a filter cake which was washed with three 5-gallon portions of benzene. The filtrate and washing were then combined and distilled under reduced pressure. There was obtained 158 lb (74% yield of theory based on PCI3) of diisopropyl fluorophosphate, BP 62°C at 9 mm and 46°C at 5 mm. 

Binding of sarin and soman to a tyrosine residue present in blood has been observed by Black et al. (51) When sarin or soman was incubated with human plasma, phosphonylated tyrosine was observed by LC/MS after Pronase digestion, in addition to phosphonylated serine. The precise site of this residue has not yet been confirmed but it is associated with the albumin fraction. A phosphonylated tryptic peptide [/-PrO(CH3)P(0)]-Tyr-Thr-Lys, consistent with albumin, has been identified but this sequence is also present in other proteins. Before the advent of modem mass spectrometry, diisopropyl fluorophosphate was reported to bind... 

Koster R. Synergism and antagonism between physostigmine and diisopropyl fluorophosphate in cats. /. Pharmacol. Exp. Then, 1946, 88, 39-46. 

The nerve gas diisopropyl fluorophosphate (DFP) reacts with the serine —OH in some enzymes to form HF and the (9-phosphoryl ester as follows ... 

Bushnell, P.J., Padilla, S.S., Ward, T., Pope, C.N., Olszyk, V.B. (1991). Behavioral and neurochemical changes in rats dosed repeatedly with diisopropyl fluorophosphate. J. Pharmacol. Exp. Ther. 256 741-50. 

Misulis, K.E., Clinton, M.E., Dettbam, W-D., Gupta, R.C. (1987). Differences in central and peripheral neural actions between soman and diisopropyl fluorophosphate, organophosphoms... 

Smallridge, R.C., Carr, F.E., Fein, H.G. (1991). Diisopropyl-fluorophosphate (DFP) reduces serum prolactin, thyrotropin, luteinizing hormone, and growth hormone and increases adrenocorticotropin and corticosterone in rats involvement of dopaminergic and somatostatinergic as well as cholinergic pathways. Toxicol. Appl. Pharmacol. 108 284—95. 

Aeylpeptide hydrolase is a member of the serine hydrolase family. It deaeetylates the acetylated N-terminus of polypeptides. Rat brain aeylpeptide hydrolase was inhibited 93% at a dose of diehlorvos (4mg/kg, i.p.) which inhibited aeetyleholinesterase only 47%. The in vitro sensitivity of aeylpeptide hydrolase to chlorpyrifosmethyl oxon, diehlorvos, and diisopropyl fluorophosphate (IC50) was 6-10 times greater than that of acetylcholinesterase (Riehards et al, 2000). Aeylpeptide hydrolase is also found in human erythroeytes where it could potentially serve as a biomarker for low dose exposure to OP in humans, though human eases of OP exposure have not yet been tested for OP-modified acylpeptide hydrolase. 

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