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Identification of Essential Carboxyl Groups in the Specific Binding Site of Bovine Trypsin by Chemical Modification

From the ¹ From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37203

The carboxyl groups essential for the enzyme activity of bovine trypsin were labeled and identified by means of a chemical modification procedure. Chromatographically purified bovine ß-trypsin, which contains a single polypeptide chain, was treated with the water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide, and glycinamide, according to the method of Koshland et al. (13–15). Coupling of the protein carboxyl groups with glycinamide in amide linkage resulted in nearly complete loss of the enzyme activity as determined by an esterolytic assay using N-benzoyl-l-arginine ethyl ester as substrate. The extent of modification of the carboxyl groups and the extent of enzyme inactivation were found to be decreased in the presence of the competitive inhibitor benzamidine, indicating that certain essential carboxyl groups were protected from modification by the inhibitor. The carboxyl groups masked by the inhibitor were then reacted, after removal of the inhibitor by dialysis, with the carbodiimide reagent and ¹⁴C-glycinamide. After enzymatic degradation of the radioactive trypsin derivative, the labeled peptides were isolated and identified by their amino acid composition. The primary site of labeling was found to be aspartyl-177, strongly suggesting that this residue provides the anionic center for specific substrate binding. Some radioactivity was also associated with Asp-182. This residue reacted very slowly with the carbodiimide reagent, but it could be readily modified in the presence of 8 m urea, indicating that it is partially buried in the molecule. In 8 m urea, another carboxyl group, Asp-90, became available to modification. The failure of this residue to react in the native state suggests that Asp-90, which is homologous with an aspartyl residue known to be buried in chymotrypsin (21), is completely buried in the trypsin molecule. The enzyme which had been modified in the absence of benzamidine and had lost 99% of its specific esterolytic activity retained its reactivity toward diisopropyl phosphorofluoridate to a significant extent, indicating that the inactivation was due primarily to loss of the specificity site. The reaction of tyrosine with the carbodiimide was not a major cause of the inactivation because the regeneration of tyrosyl residues by hydroxylamine resulted in no significant change in the activity of modified trypsin preparations.

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