Diisopropyl fluorophosphate inhibited chymotrypsin

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). 

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. 

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 mechanism of the action of acetylcholinesterase purified from the electric organs of Electrophorus electricus involves the attraction of the positively charged nitrogen of acetylcholine to an anionic site on the enzyme and cleavage of the substrate at an esteratic site of a nucleophilic character. The irreversible inhibition by the alkyl phosphates, tetraethyl pyrophosphate (TEPP) and diisopropyl-fluorophosphate (DFP) may be due to phosphorylation of the nucleophilic esteratic site. The phosphorylation by DFP of the phenolic hydroxyl group of free tyrosine has been demonstrated by Ashbolt and Rydon. Chymotrypsin and citrus fruit acetylesterase are also inhibited by DFP and TEPP. ... 

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