Reaction of human butyrylchounesterase (BCHE) His-117 enzymes with carbamates

By replacing a Gly in the "oxyanion hole" of BChE with His, we designed two enzymes (Gl I7H and Gl I7HEI97Q) that hydrolyze organophosphorus nerve agents and related covalent inhibitors. This hydrolytic aaivity is not observed with wildtype (WT) BChE, Gl I7K, or EI97Q (Biochemistry 34:15295,1995; 36:786-795, 1997). Carbamates (CBs) also inhibit BChE via a covalent intermediate. We report here on the reaction of the His-l 17 enzymes with two CBs, using butyrylthiocholine (BTCh) as substrate at pH 7.5,25 C: Physostigmine (phy) Pyridostigmine (pyr) k, (H'W) kj(min') k,(M'minJ) k,(min') WTBChE 1,050,000 0.0060 10,000 0.0024 GII7H 13,600 0.0018 250 0.0009 GII7K 14.800 0.0090 200 ND GII7HEI97Q 14 0.0005 60 0.0008 Carbamylation of Gl I7H or Gl I7K is much slower than WT. This may result from steric hindrance (phy) or charge repulsion (pyr) in the reversible complex. In contrast, decarbamylation (kj decreases only 2- to 3-fold for Gl I7H and actually increases for Gl I7K compared to WT. This observation is consistent with the 2.5-fold reduction in BTCh k-4 previously reported for GII7H. In contrast to the organophosphorus inhibitors, stabilization of the tetrahedral transition states of CBs or carbonyl esters (BTCh) appears not to be severely impeded by the bulky His or Lys side chain. Also, the decrease of > 10-fold in k, for GII7HEI97Q inhibited with phy, but not Gl I7H, provides evidence that EI97 can influence cholinesterase decarbamylation.