Fluorophosphate esters, hydrolysis

It is found that the hydrolysis of fluorophosphate esters is also accelerated by transition metal ions and complexes. This would be an observation of little general interest, except for the fact that fluorophosphate esters form one of the more commonly encountered types of nerve gases (Fig. 4-51). The hydrolysis of fluorophosphate esters is increased dramatically in the presence of copper(n) and other transition metal complexes, and this sug-... 

The formation of copper(II) complexes with terpy has been investigated fairly intensively. The interaction is pH dependent, and numerous hydroxy, aqua, and polynuclear species are present in aqueous solution 94, 245,278). In general, an Eigen-Wilkins mechanism appears to be operative, although the kinetics are complicated by ligand-protonation equilibria 263,390,391). In acidic solution, 1 1 complexes predominate (567). A number of substituted terpyridine ligands have been evaluated as potential colorimetric reagents for copper 400). The adsorption behavior of copper(II)-terpy complexes at silica surfaces has been studied 499). Such complexes are reasonably active as catalysts for the hydrolysis of fluorophosphate esters 456). 

Ligands closely related to orthophosphate esters include acetylphosphate, acetylphenylphosphate and fluorophosphate. Alkaline hydrolysis of [Co OP(0)20COMe (NH3)5] occurs exclusively at the carbonyl centre, and the acceleration provided by cobalt(III) four atoms removed is minimal (10 times, equation This is a good comparative example of phosphoryl versus acyl hydrolysis,... 

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. 

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. 

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