Averell Gnatt

Excavations into the active-site gorge of cholinesterases

Acetyl- and butyrylcholinesterase (ACHE, BCHE) from evolutionarily distant species display a high degree of primary sequence homology and have biochemically similar catalytic properties, yet they differ in substrate specificity and affinity for various inhibitors. The biochemical information derived from analyses of ACHE and BCHE from human, Torpedo, mouse, and Drosophila, as well as that from the recombinant forms of their natural variants and site-directed mutants, can currently be re-examined in view of the recent X-ray crystallography data revealing the three-dimensional structure of Torpedo ACHE. The picture that emerges deepens the insight into the biochemical basis for choline ester catalysis and the complex mechanism of interaction between cholinesterases and their numerous ligands.

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes

Summary1.Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities.2.Homologous cosmid recombination with a 190-base pair 5′ fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3′ end of BuChEcDNA from nervous system tumors and which has been mapped byin situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns.3.The chromosomal origin of BuChE-coding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringentin situ hybridization protocol, including washes at high temperature and low salt.4.These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family of highly similar proteins encoded by distinct, nonhomologous genes.

Site-Directed Mutagenesis of Active Site Residues Reveals Plasticity of Human Butyrylcholinesterase in Substrate and Inhibitor Interactions

Abstract: 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 Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso‐OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10‐fold decreased IC50 values for DFP, iso‐OMPA, and echothiophate. These findings add Tyr440 and Phe329 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.

Molecular biological search for human genes encoding cholinesterases.

Cholinesterases (ChEs) are highly polymorphic proteins, capable of rapidly hydrolyzing the neurotransmitter acetylcholine and involved in terminating neurotransmission in neuromuscular junctions and cholinergic synapses. In an attempt to delineate the structure and detailed properties of the human protein(s) and the gene(s) coding for the acetycholine hydrolyzing enzymes, a human cDNA coding for ChE was isolated by use of oligodeoxynucleotide screening of cDNA libraries. For this purpose, a method for increasing the effectiveness of oligonucleotide screening by introducing deoxynosine in sites of codon ambiguity and using tetramethyl-ammonium salt washes to remove false-positive hybrids was employed. The resulting isolated 2.4-kilobase (kb) cholinesterase cDNA sequences encode for the entire mature secretory protein, preceded by an N-terminal signal peptide. The human ChE primary sequence shows almost no homology to other serine hydrolases, with the exception of a hexapeptide at the active site. In contrast, it displays extensive homology with acetycholinesterase formTorpedo californica andDrosophila melanogaster as well as with bovine thyroglobulin. These extensive homologies probably suggest the need of the entire coding sequence for the physiological function(s) fulfilled by the enzyme and further suggest a common, unique, ancestral gene for these cDNAs. In turn, the cDNA was used as a probe to isolate genomic DNA sequences for the 5′-region of the human ChE gene. The genomic DNA fragment encoding part of the 5′-region of ChEcDNA was detected by DNA blot hybridization, enriched 70-fold by gel electrophoresis and electroelution, cloned in λ phage and isolated. Sequencing of the cloned DNA revealed that it did indeed include part of the 5′-region of ChEcDNA, starting at an adjacent 5′-position to the nucleotides coding for the initiator methionine, and ending with anEcoRI restriction site inherent to the ChEcDNA sequence. The isolated fragment of the human cholinesterase gene is currently employed to complete the structural characterization of this and related genes.

Testicular Gene Amplification and Impaired BCHE Transcription Induced in Transgenic Mice by the Human BCHE Coding Sequence

Multiple findings implicate acetylcholine with sperm functioning 1,2 and acetyl-and butyrylcholinesterase activities (ACHE, BCHE) were observed in mammalian sperm cells and during oocyte development 1–3. In vivo amplification of the human BCHE gene was first found in a father and son exposed to cholinesterase inhibitors 4, but it remained unclear whether the amplified DNA was transmitted as such from father to son or whether the amplification phenomenon re-occurred in germ cells, particularly during male meiosis or sperm differentiation.

Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses.

1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. Thus, it was found that asp70-->gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425-->Pro with gly70 gives rise to the atypical butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimers and Parkinsons disease, glaucoma and myasthenia gravis.

Molecular Dissection of Functional Domains in Human Cholinesterases Expressed in Microinjected Xenopus Oocytes

The two classes of human cholinesterases (CHEs), acetylcholinesterase(acetylcholine acetyl hydrolase, ACHE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acyl hydrolase, BCHE, EC 3.1.1.8) are highly homologous proteins capable of rapidly hydrolyzing choline estersl. Despite their similar mechanisms of action, they differ in substrate specificity and sensitivity to various inhibitors1,2. Recent advances including cloning,3–6 expression,5,7–10 and 3 dimensional structural analysis of members of the CHE superfamily,11,12 now enable the dissection of functional domains thereof. Within these domains, key amino acids are found which may be implicated in catalysis or in binding of various ligands. The disclosing of such key residues could lead to designing of novel therapeutic agents as well as to the unravelling of the molecular mechanisms underlying the functioning of ChEs.

Structure-Function Relationships, In Vivo Mutability and Gene Amplification in Human Cholinesterases, Targets for Organophosphorous Poisons

The human Cholinesterase genes and their protein products have been the focus of intensive research for many years (for comprehensive reviews see Whittaker, 1986; 1Rakonczay and Brimijoin, 1988; and Soreq and Zakut, 1990) because of the physiological function attributed to these enzymes, which are both capable of hydrolyzing the neurotransmitter acetylcholine. Genetic linkage evidence indicated that two distinct genes, designated ACHE and CHE, encode the two principal forms of cholinesterases, acetylcholinesterase (acetylcholine acetyl hydrolase, AChE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, BuChE, EC 3.1.1.8) which differ in their substrate specificities and sensitivities to selective inhibitors. The toxic effects of organophosphorous (OP) poisons, such as common insecticides or nerve gases, are generally attributed to their specific inhibition of cholinesterases, interfering with cholinergic neurotransmission. OP inhibition of cholinesterases occurs through a covalent interaction of the OP compounds with a serine residue in the active esteratic site (Koelle, 1972). However, detailed structure-function relationships in this family of enzymes have been hampered by the difficulties in purifying mammalian cholinesterases.

STRUCTURE-FUNCTION RELATIONSHIP STUDIES IN HUMAN CHOLINESTERASES AS AN APPROACH FOR EVALUATING POTENTIAL PHARMACOTHERAPEUTIC AND/OR TOXICITY EFFECTS OF CHOLINERGIC DRUGS

Attempts to restore cholinergic deficits in Alzheimer’s disease patients involve the use of various drugs inhibiting cholinesterases (CHEs; Bartus et al., 1982). In addition, CHE inhibitors are clinically employed in the treatment of other common syndromes, including Parkinson’s disease, myasthenia gravis and multiple sclerosis (Taylor, 1990). The efficacy and specificity of such drugs on the one hand, and their toxicity factor on the other, largely depend on their inhibitory effects on CHEs in the treated individuals. Therefore, updated evaluation methods for these parameters should be valuable to pharmacological research focused on the development and use of cholinergic drugs.