HISTIDINE MODIFICATION OF HUMAN SERUM BUTYRYLCHOLINESTERASE

The effects of histidine-modifying reagents on human serum butyrylchol inesterase (BChE) were investigated, The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column pr ior to use, In the modification studies, we found that the histidine-s pecific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tos yllysine chloromethyl ketone (TLCK) did not modify the enzyme; however , they inhibited the enzyme reversibly. The kinetic parameters of enzy me inhibition calculated were alpha = 10.8, beta = 0.26, and K-i = 0.0 16 mM for TPCK. TLCK inhibition gave similar kinetic behavior, with al pha = 41.6, beta = 0.065, and K-i = 0.039 mM. Tosyllysine, an analog o f TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialys is resulted in significant reactivation of the enzyme. From kinetic st udies, it was found that the inhibitions were hyperbolic mixed-type in hibitions. 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 (DP C), it was observed that inactivation of the enzyme was irreversible a nd time-dependent. In the protection studies, the activity of the enzy me was partially protected from inactivation by DPC even at a 50 mM co ncentration of butyrylthiocholine. The results indicate that DPC modif ies same essential histidine side chains in BChE, including the functi onal histidyl residue found at the active site. (C) 1997 Academic Pres s.