13C NMR study of how the oxyanion pKa values of subtilisin and chymotrypsin tetrahedral adducts are affected by different amino acid residues binding in enzyme subsites S1-S4.

A range of substrate-derived chloromethane inhibitors have been synthesized which have one to four amino acid residues. These have been used to inhibit both subtilisin and chymotrypsin. Using 13C NMR, we have shown that all except one of these inhibitors forms a tetrahedral adduct with chymotrypsin, subtilisin, and trypsin. From the pH-dependent changes in the chemical shift of the hemiketal carbon of the tetrahedral adduct, we are able to determine the oxyanion pKa in the different inhibitor derivatives. Our results suggest that in both the subtilisin and chymotrypsin chloromethane derivatives the oxyanion pKa is largely determined by the type of amino acid residue occupying the S1, subsite while binding in the S2-S4 subsites only has minor effects on oxyanion pKa values. Using free energy relationships, we determine that the different R groups of the amino acid residues binding in the S1 subsite only have minor effects on the oxyanion pKa values. We propose that the lower polarity of the chymotrypsin active site relative to that of the subtilisin active site explains why the oxyanion pKa is higher and more sensitive to the type of chloromethane inhibitor used in the chymotrypsin derivatives than in the subtilisin derivatives.

A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct.

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane++ + and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3-103.6 p.p.m., depending on the pH. The titration shift of 4.7-4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR.

Subtilisin BPN' has been alkylated using benzyloxycarbonyl-glycylglycyl[1-13C]phenylalanylchloromethane+ ++. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched alpha-methylene carbon of the subtilisin-(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/ autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the alpha-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin-(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin-(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.

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.