Studies with Potential Reporter Group Reagents for Enzymes: 3,4-Dihydro-3-(2-Hydroxyethyl)-6-Nitro-2H-1,3-Benzothiazin-2-Thione and 6-Nitrochromone—Hydrolysis and Interaction with Chymotrypsin

Abstract

3,4-Dihydro-3-(2-hydroxyethyl)-6-nitro-2H-1,3-benzothiazin-2-thione (II-HE), a cyclic dithiocarbamate, has the potential to react with an esterase to furnish it with a p-nitrobenzenethiolate “reporter group.” However, unlike a closely similar cyclic carbamate, II-HE is totally without reaction on chymotrypsin. Possible reasons for this major effect of substituting sulfur for oxygen are discussed. The results support the idea that chymotrypsin's “oxyanion hole” cannot properly accommodate a thioanion. II-HE undergoes an interesting intramolecular cleavage reaction under alkaline conditions. The mechanism of this process has been determined using evidence from NMR and mass spectrometry. 6-Nitrochromone (6-NC) likewise has the potential to modify an enzyme covalently and thereby act as a reporter group reagent. With chymotrypsin, 6-NC reacts as predicted, except that the attached label is stable only at high pH; the labeling reaction slowly reverses at low pH. From the lack of effect on enzyme activity, it is clear that modification of chymotrypsin by 6-NC does not occur at the active site.