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 a nd inhibitor specificities of butyrylcholinesterase (BuChE), we employ ed site-directed mutagenesis to modify the catalytic triad residue Ser (198), the acyl pocket Leu(286) and adjacent Phe(329) residues, and Me t(437) and Tyr(440) located near the choline binding site. Mutant prot eins were produced in microinjected Xenopus oocytes, and K-m values to wards butyrylthiocholine and IC50 values for the organophosphates diis opropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide ( echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were de termined. Substitution of Ser(198) by cysteine and Met(437) by asparta te nearly abolished activity, and other mutations of Ser(198) complete ly abolished it. Tyr(440) and Leu(286) mutants remained active, but wi th 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 IC 50 Values increased for Leu(286) mutants in the order Asp < Gin < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe(329) dis played unmodified K-m values toward butyrylthiocholine, but up to 10-f old 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.