DETERMINATION OF THE IONIZATION STATE OF THE ACTIVE-SITE HISTIDINE INA SUBTILISIN-(CHLOROMETHANE INHIBITOR) DERIVATIVE BY C-13-NMR

Subtilisin BPN' has been alkylated using nyl-glycylglycyl[1-C-13]pheny lalanylchloromethane. Using difference C-13-NMR spectroscopy a single signal due to the C-13-enriched alpha-methylene carbon of the subtilis in-(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 o f SDS at pH 6.9 did denature this derivative. The negative titration s hift of the alpha-methylene carbon of the denatured derivatives confir med 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 pK(a) of 7.04 obtained from studying the native subtilisin-(chlor omethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pK(a) of the alkylated histidine residue in the nati ve subtilisin-(chloromethane inhibitor) derivative must be > 12 and th at subtilisin preferentially stabilizes the zwitterionic tetrahedral a dduct consisting of the oxyanion and the imidazolium ion of the active -site histidine residue. We show that even before the oxyanion is form ed the pK(a) of the active-site histidine must be much greater than th at of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the ser ine proteinases.