SITE-DIRECTED MUTAGENESIS OF ACTIVE-SITE RESIDUES REVEALS PLASTICITY OF HUMAN BUTYRYLCHOLINESTERASE IN SUBSTRATE AND INHIBITOR INTERACTIONS

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser(198), the acyl pocket Leu(286) and adjacent Phe(329) residues, and Met(437) and Tyr(440) located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and K-m values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser(198) by cysteine and Met(437) by aspartate nearly abolished activity, and other mutations of Ser(198) completely abolished it. Tyr(440) and Leu(286) mutants remained active, but with higher K-m and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu(286) mutants. Both K-m and IC50 Values increased for Leu(286) mutants in the order Asp < Gin < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe(329) displayed unmodified K-m values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Try(440) and Phe(329) to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.