A STUDY OF THE STABILIZATION OF THE OXYANION OF TETRAHEDRAL ADDUCTS BY TRYPSIN, CHYMOTRYPSIN AND SUBTILISIN

Subtilisin and S-chymotrypsin have been alkylated using 2-C-13-enriche d oxycarbonylglycylglycylphenylalanylchloro-methane. A single signal d ue to the C-13-enriched carbon was detected in both the intact subtili sin and delta-chymotrypsin derivatives. The signal titrated from 98.9 p.p.m, to 103.6 p.p.m. with a pK(a) value of 6.9 in the subtilisin der ivative and it is assigned to a tetrahedral adduct formed between the hydroxy group of serine-221 and the inhibitor. The signal in the delta -chymotrypsin derivative titrated from 98.5 p.p.m. to 103.2 p.p.m. wit h a pK(a) value of 8.92 and it is assigned to a tetrahedral adduct for med between the hydroxy group of serine-195 and the inhibitor. In both derivatives the titration shift is assigned to the formation of the o xyanion of the tetrahedral adduct. delta-Chymotrypsin has been inhibit ed by benzyloxycarbonylphenylalanylchloromethane and two signals due t o C-13-enriched carbons were detected. One of these signals titrated f rom 98.8 p.p.m. to 103.6 p.p.m. with a pK(a) value of 9.4 and it was a ssigned in the same way as in the previous delta-chymotrypsin derivati ve. The second signal had a chemical shift of 204.5 +/- 0.5 p.p.m. and it did not titrate from pH 3.5 to 9.0. This signal was assigned to al kylated methionine-192. We discuss how subtilisin and chymotrypsin cou ld stabilize the oxyanion of tetrahedral adducts.