Phosphorus phosphoryl serine

The reaction of an OP with AChE, BuChE, or other B-esterases is similar to the reaction of AChE with ACh, except that the hydrolysis step is much slower or, in some cases, may not occur at all. Its basis is a phosphorylation of the enzyme via a nucleophilic attack. The electronegative serine hydroxyl at the catalytic site reacts with the electropositive phosphorus atom of the inhibitor to form an OP-ChE complex and loss of a side group on the phosphorus atom, known as the leaving group (X). The phosphorylated enzyme may, in time, reactivate by... 

Many pesticides are esters or amides that can be activated or inactivated by hydrolysis. The enzymes that catalyze the hydrolysis of pesticides that are esters or amides are esterases and amidases. These enzymes have the amino acid serine or cysteine in the active site. The catalytic process involves a transient acylation of the OH or SH group in serin or cystein. The organo-phosphorus and carbamate insecticides acylate OH groups irreversibly and thus inhibit a number of hydrolases, although many phosphorylated or carbamoylated esterases are deacylated very quickly, and so serve as hydrolytic enzymes for these compounds. An enzyme called arylesterase splits paraoxon into 4-nitrophenol and diethyl-phosphate. This enzyme has cysteine in the active site and is inhibited by mercury(ll) salts. Arylesterase is present in human plasma and is important to reduce the toxicity of paraoxon that nevertheless is very toxic. A paraoxon-splitting enzyme is also abundant in earthworms and probably contributes to paraoxon s low earthworm toxicity. Malathion has low mammalian toxicity because a carboxyl esterase that can use malathion as a substrate is abundant in the mammalian liver. It is not present in insects, and this is the reason for the favorable selectivity index of this pesticide. 

The phosphorus ester molecule, blocking the activity of acetylcholinesterase, first takes up a steric orientation determined by the anionic and acidic groups of the enzyme, and this is then followed by hydrolysis of the phosphorus ester and by phosphorylation of the hydroxyl group of serine at the esteratic site of the enzyme ... 

In the first type of mechanism, a nucleophile in the enzyme (analogous to serine 195 in chymotrypsin) attacks the phosphoryl group to form a covalent intermediate. In a second step, this intermediate is hydrolyzed to produce the final products. Because two displacement reactions take place at the phosphorus atom in the first mechanism, the stereochemical configuration at the phosphorus atom would be inverted and then inverted again, and the overall configuration would be retained. 

Chymotrypsin, in addition to its proteolytic activity, can also function as an esterase. It is inactivated by D.F.P., etc. (p. 186). The esterases firmly bind the phosphorus of D.F.P., and in the case of chymotrypsin the reaction is bimolecular, yielding a crystalline derivative containing two isopropoxy groups and one atom of phosphorus per protein molecule, but no fiuorine. Recently the compound of chymotrypsin and labelled D.F.P. has been hydrolysed and the hydrolysate shown to contain L-serine phosphoric acid. The latter is known to be phosphoryl-ated on its hydroxyl group. ... 

PhOSphorylatGd Proteins. Naturally occurring phosphorus-containing proteins include such important examples as casein, discussed elsewhere in this Encyclopedia. In living organisms, proteins are often phosphorylated enzymatically on the serine or tyrosine hydroxyl group as an activation or regulation step, and over-phosphorylation may play a role in disease this biochemistry is outside the scope of the present article. 

Careful analysis of the linewidth of the E P complex has indicated that the dissociation of noncovalent phosphate is the rate-Umiting step in the turnover of the enzyme (Hull et al., 1976). A study of the phosphorylated apoenzyme revealed that the linewidth observed was much narrower than that for the metal-containing enzyme. This suggested considerable flexibility for the residue (Chlehowski et al, 1976) in contrast to the holoenzyme, where the observed linewidth has been interpreted as that of a rigidly held moiety (Vogel et al., 1982). pH titrations showed that the residue in the apoenzyme cannot be protonated until the enzyme dissociates and unfolds at low pH (Chlehowski et al., 1976). The phosphorus nucleus in the apo-phosphoryl enzyme is coupled to the methylene protons of the serine with a coupling constant of 13 Hz, indicating that rotomers 1 or 3 are dominant and not much rotomer 2 is present, in contrast to the model compound (see 3, Scheme 3) (Chlehowski et al., 1976). 

Section IV,B,2 introduces the idea that chemical-shift anisotropy could become an important relaxation mechanism for phosphorus at hi field. Vogel et al. (1982) have published the field dependence of the linewidth of the histidine 3-iV-phosphate of succinyl-CoA synthetase and the serine phosphate of glycogen phosphorylase a. They found that the CSA mechanism did, indeed, dominate at 6.3- and 9.3-T fields, contributed substantially at 4.7 T, and contributed about 25% of the total relaxation at 2.1 T (36.4 MHz). Vogel etal. 1982) estimated for Escherichia coli sucdnyl-CoA synthetase (MW 140,(XX)) a hydrated radius of 37 A and an isotropic correlation time of 44 ns. These numbers are referenced to the phosphorus linewidth (sensitive only to overall motion) using an analysis very similar to that mentioned in Section IV,B on DNA phosphorus relaxation. In contrast, the E. coli phosphoryl carrier protein HPr (MW 9(KX)) has a calculated hydrated radius of 17 A and a correlation time of 4.2 ns, according to Vogel et al. (1982). 

Antidotes. Because the acute toxicity to man of many of the phosphorus insecticides is high, first-aid remedies are kept on hand. The most useful of these is an injection of atropine (which acts primarily on muscarinic sites) followed by an oxime specific for nicotinic sites. These oximes reactivate phosphorylated acetylcholinesterase in patients (Holmes and Robins, 1955) just as with the isolated enzyme (Wilson and Meislich, 1953). This reaction involves a competition, between the hydroxyl-groups of serine and of the hydroxylamine, for the phsphoryl-group. The covalent bond with the serine is broken, and simultaneously a new covalent bond formed with the hydroxylamine (see Scheme 12.1). One of the best reactivators is pralidoxime 12.27) (2-PAM), which is the anti form of pyridine-2-aldoxime methochloride. Widely differing doses of antidote are required, depending on the strength of the phosphate-enzyme bond, which varies with the nature of the insecticide. 

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