Mutagenesis of essential functional residues in acetylcholinesterase.

Abstract

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 assignnment 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 ofa 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 substrateinhibition 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.Efficiently catalyzed hydrolysis of the neurotransmitter acetylcholine is critical to proper functioning of the cholinergic nervous system, and several pharmacologic and toxicologic agents act by inhibiting acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7). AChE functions as a serine hydrolase (1-3) and enzyme inhibitors used clinically or as insecticides typically serve as alternative substrates by acylating the active-center serine with slower leaving groups. The primary structure of AChE revealed that it lacks global sequence similarities with serine hydrolases of the trypsin and subtilisin families and only possesses residue identity with trypsin immediately around the active-center serine (4). Surprisingly, AChE is homologous to the carboxyl-terminal domain of a large secreted glycoprotein, thyroglobulin, the precursor to thyroid hormone (4). Subsequent primary structures showed that the cholinesterases belong to a distinct, but functionally eclectic, family of serine hydrolases. Included in this family are the butyrylcholinesterases, hepatic microsomal carboxylesterases, the Drosophila Est-6, lysophospholipases, cholesterol esterases, and two proteins found in inclusion bodies of Dictyostelium (5-11).The cholinesterases are characterized by their specificity for choline esters but can be subdivided on the basis of acyl group selectivity. The true AChEs (EC 3.1.1.7) show weak catalytic activity for choline esters with acyl groups larger than propionylcholine; the butyrylcholinesterases (EC 3.1.1.8) efficiently hydrolyze esters with large...

Structural modifications of the Ω loop in human acetylcholinesterase

“…In a preliminary experiment, designed to test whether the proper positioning of Trp-86 in the active center is the only catalytically significant function of the Lb3,2 loop, deletion of [3 Trp/Phe-86 most of its mobile part was attempted. However, attempts to express a protein, carrying excision of the sequence Asp-74-.…”

“…Residue Trp-86 catalytic activity and in aliosterism, which in its active conformation is the main element of the classical 'anionic subsite ' [6,10,11,26,27] ). In the case of lipases, X-ray crystallographic together with kinetic studies of the AChE muteins with substudies of free versus complexed structures show that the Lba,2 strates and reversible inhibitors [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. However, the location loop is one of the most mobile structural elements [18,22,32].…”

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

“…The active-site serine has been mapped to position 200 in the enzyme from Torpedo electric organ (21). The histidine residue involved in catalysis has been shown to be His-440 (22). Especially the sequence around the active-site serine is highly conserved in all enzymes mentioned above.…”

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

“…hydrolases generally resulted in inactive proteins, consistent with the role of this residue as an essential catalytic base (4,9,17,24,35,44,53,56,65). However, reports on protein variants of C-C hydrolase MhpC (40) and Agrobacterium radiobacter epoxide hydrolase (59) show that the observation of minor residual activity after replacement of the triad's histidine is not unprecedented.…”