MODIFICATION OF ESSENTIAL CARBOXYLIC ACID SIDE CHAINS OF TRYPSIN

In attempts to chemically modify carboxyl groups of importance for the function of trypsin, particularly one(s) presumably responsible for its specificity, conditions of reaction with isoxazolium salts were studied. Both the structure of the reagent and the pH of the modification reaction were important in achieving a selective modification. N-Methyl-5-phenylisoxazolium fluoroborate and N-ethyl-5-phenylisoxazolium at pH 3.8 resulted in the most specific reaction, producing nearly complete inactivation by the modification of two to three carboxyl groups. This effect was largely prevented when the reaction was carried out in the presence of a competitive inhibitor, benzamidine. The enol esters produced by activation with isoxazolium salts were treated with glycine ethyl ester and O-methylhydroxylamine, forming the corresponding amides in high yields. This second change did not affect the degree of inactivation. N-Methylhydroxylamine, on the other hand, although fully displacing the activating reagent, was not incorporated and restored tryptic activity. Hydroxylamine also quantitatively displaced the reagent with some reactivation, the extent of which varied with reaction time and degree of initial modification. Possible explanations for these phenomena are discussed. Lessen degradation of the trypsin hydroxamic acid derivative indicated modification of both aspartic and glutamic acid residues had taken place. The observation that diisopropyl phosphorofluoridate reacted to a greater extent than 1-chloro-3-tosylamido-7-amino-2-heptanone with several modified trypsin derivatives indicated the existence of enzyme species with altered enzymatic activity in the product mixture. At least two stages of functional change were produced by carboxyl modification affecting specificity and serine reactivity in that order. © 1969, American Chemical Society. All rights reserved.