THE inhibition of chymotrypsin by diethyl p-nitrophenyl phosphate.

The alkyl fluorophosphonates were first shown in 1940 (Adrian, Feldberg & Kilby, 1947) to be very powerful inhibitors of cholinesterase, and for a time they were thought to be quite specific for this enzyme. Later, other esterases, such as human milk lipase and liver esterase (Webb, 1948) and citrus acetylesterase (Jansen, Nutting & Balls, 1947) were also shown to be inhibited, although not to the same extent. The discovery that trypsin and chymotrypsin display esterase as well as proteolytic activity (Schwert, Neurath, Kaufman & Snoke, 1948; Kaufman, Schwert & Neurath, 1948) prompted the investigation of the action of fluorophosphonates on these enzymes (Jansen, Nutting, Jang & Balls, 1949), and it was found that both were sensitive to diiwopropyl fluorophosphonate (DFP) and that the esterase and proteolytic activities were inhibited in parallel. A considerable number of other organic phosphorus compounds have been discovered which resemble the fluorophosphonates in being highly toxic and powerful anti-cholinesterases. Some of these are now being produced commercially as insecticides, OO-diethyl 0-p-nitrophenyl thiophosphate (Parathion) being one of the best known. The increasing use of such toxic compounds makes a fuller knowledge of their mode of action desirable. It would also be valuable if one could eventually suggest, on a rational basis, the direction in which a search could most profitably be made for compounds of high insecticidal activity combined with low mammalian toxicity. It appeared to us that a suitable starting point for the investigation of the mode of action of organic phosphorus compounds would be a detailed study of a model system using an inhibitor whose chemistry is straightforward and an enzyme which could be obtained pure and crystalline, preferably of small and known molecular weight. As an inhibitor, we have selected diethyl p-nitrophenyl phosphate (E 600, Paraoxon), the oxygen analogue of Parathion, as the chemistry of the latter is complicated by isomerization and really pure material is very difficult to obtain. Chymotrypsin has been found to be a very suitable enzyme, and is interesting in possessing several types of enzymic activity. The enzymic hydrolysis of certain p-nitrophenyl esters has been investigated, and the inhibition of the proteolytic, esterase and amidase activities of chymotrypsin by E 600 has been studied.