A study of the stabilization of the oxyanion of tetrahedral adducts by trypsin, chymotrypsin and subtilisin.
Subtilisin and delta-chymotrypsin have been alkylated using 2-13C-enriched benzyloxycarbonylglycylglycylphenylalanylchloromethane. A single signal due to the 13C-enriched carbon was detected in both the intact subtilisin and delta-chymotrypsin derivatives. The signal titrated from 98.9 p.p.m. to 103.6 p.p.m. with a pKa value of 6.9 in the subtilisin derivative 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. with a pKa value of 8.92 and it is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. In both derivatives the titration shift is assigned to the formation of the oxyanion of the tetrahedral adduct. delta-Chymotrypsin has been inhibited by benzyloxycarbonylphenylalanylchloromethane and two signals due to 13C-enriched carbons were detected. One of these signals titrated from 98.8 p.p.m. to 103.6 p.p.m. with a pKa value of 9.4 and it was assigned in the same way as in the previous delta-chymotrypsin derivative. 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 alkylated methionine-192. We discuss how subtilisin and chymotrypsin could stabilize the oxyanion of tetrahedral adducts.
O'Connell
T P
TP
Department of Biochemistry, University College Dublin, Ireland.
Malthouse
J P
JP
D000595:Amino Acid Sequence; D000838:Anions; D002247:Carbon Isotopes; D002918:Chymotrypsin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
D000838:Anions; D002247:Carbon Isotopes; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin