Cengiz_1997_Biochem.Mol.Med_61_52

The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCE) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were alpha = 10.8, beta = 0.26, and Ki = 0.016 mM for TPCK. TLCK inhibition gave similar kinetic behavior, with alpha = 41.6, beta = 0.065, and Ki = 0.039 mM. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mM concentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.