Mutagenesis of essential functional residues in acetylcholinesterase.
The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.
Gibney
G
G
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Camp
S
S
Dionne
M
M
MacPhee-Quigley
K
K
Taylor
P
P
D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics; D014101:Torpedo; D014162:Transfection
D002800:Cholinesterase Inhibitors; D015345:Oligonucleotide Probes; D000110:Acetylcholinesterase; D012697:Serine Endopeptidases