10] TRYPSIN AND ORG ANOPHOSPHORUS COMPOUNDS 177 



It has been found that the incorporation of the first phosphate on the enzyme 

 is a rapid reaction and leads to inactivation. The second phosphate is intro- 

 duced at a much slower rate than the first. 



E600 under identical conditions yields only 1-0 mole phosphorus per 

 mole of the enzyme. It was noted that the reaction between trypsin and 

 E600 occurs at a much lower rate compared with that with DFP. This obser- 

 vation might account for the differences in the phosphorus incorporation 

 data. 



(III) Blocking the amino groups of trypsin by acetylation (73% of amino 

 groups blocked as shown by ninhydrin determination) impairs the ability 

 of the enzyme to react with E600 in the presence of urea, whilst the reaction 

 in aqueous medium can still occur. 



(IV) The inactive precursor, chymotrypsinogen, could be phosphorylated 

 by DFP either in aqueous medium or in the presence of urea. A polyphos- 

 phoryl derivative capable of activation is formed. 



(V) A soluble, active di/50propyl phosphoryl trypsin (with 0-75 moles 

 'P' per mole and 81% activity) has been obtained. All the above-mentioned 

 results suggest that an active enzyme is not an essential pre-requisite for the 

 reaction with DFP and E600. These results and conclusions thereof are in 

 apparent disagreement with those of Harris and Hartley' and Neurath and 

 his associates.^ The observations in the present study have been interpreted 

 along the following hnes: 



The reaction of trypsin with DFP or E600 may be visualized to proceed 

 by two independent phosphorylation mechanisms. The first one is perhaps 

 catalysed by an imidazole group (or some other group in the case of E600) 

 on the protein. This reaction is fast and leads to inactivation. The second 

 phosphorylation, which is a slow process, appears to be mediated by an 

 amino group. A common feature in both mechanisms appears to be a 

 migration of the phosphoryl moiety to an acid-stable acceptor group. Whilst 

 the first mechanism ceases to operate in strong urea solution, the latter is 

 unaffected by such changes in the medium. Acetylation appears to eliminate 

 the second mechanism. The second phosphorylation reaction has no influ- 

 ence on the enzymic activity if carried out under conditions when the first 

 one is not operative as in the presence of urea. 



With this information, efforts were next concentrated to obtain some idea 

 of the nature of the active site(s) involved in the reaction. 



The results obtained are summarized below: 



(I) Histidine or imidazole fails to catalyse the hydrolysis of E600 at pH 

 7-6, whilst DFP hydrolysis is enhanced by the presence of histidine. 



(II) Hydroxylamine is capable of enhancing the rate of DFP and E600 

 hydrolysis. 



(III) None of the amino acids shows any catalytic activity towards E600. 



(IV) Studies on the pH optimum for the inactivation of trypsin by the 

 inhibitors show that the rate of inactivation by DFP increases with pH from 



M PS 



