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Featured researches published by Weihua Xie.


Biochimica et Biophysica Acta | 1999

Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates

Patrick Masson; Weihua Xie; Marie Thérèse Froment; Vladislav Levitsky; Pierre Louis Fortier; Christine Albaret; Oksana Lockridge

Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.


Biochimica et Biophysica Acta | 2001

Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase

Patrick Masson; Weihua Xie; Marie Thérèse Froment; Oksana Lockridge

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.


Chemico-Biological Interactions | 1999

KNOCKOUT OF ONE ACETYLCHOLINESTERASE ALLELE IN THE MOUSE

Weihua Xie; Phillip J. Wilder; Judith A. Stribley; Arnaud Chatonnet; Angie Rizzino; Palmer Taylor; Steven H. Hinrichs; Oksana Lockridge

One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.


Biochemical Pharmacology | 2000

Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma

Cynthia F. Bartels; Weihua Xie; Amanda K. Miller-Lindholm; Lawrence M. Schopfer; Oksana Lockridge

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.


Chemico-Biological Interactions | 1999

Human serum paraoxonase (PON1): Identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis

Denis Josse; Weihua Xie; Patrick Masson; Oksana Lockridge

Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.


Chemico-Biological Interactions | 1999

Tryptophan residue(s) as major components of the human serum paraoxonase active site

Denis Josse; Weihua Xie; Patrick Masson; Lawrence M. Schopfer; Oksana Lockridge

Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.


Biochimica et Biophysica Acta | 1999

Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant

Vladislav Levitsky; Weihua Xie; Marie Thérèse Froment; Oksana Lockridge; Patrick Masson

Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behavior of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.


Journal of Pharmacology and Experimental Therapeutics | 2000

Postnatal Developmental Delay and Supersensitivity to Organophosphate in Gene-Targeted Mice Lacking Acetylcholinesterase

Weihua Xie; Judith A. Stribley; Arnaud Chatonnet; Phillip J. Wilder; Angie Rizzino; Rodney D. McComb; Palmer Taylor; Steven H. Hinrichs; Oksana Lockridge


Journal of Pharmacology and Experimental Therapeutics | 2001

Evidence for Nonacetylcholinesterase Targets of Organophosphorus Nerve Agent: Supersensitivity of Acetylcholinesterase Knockout Mouse to VX Lethality

Ellen G. Duysen; Bin Li; Weihua Xie; Lawrence M. Schopfer; Robert S. Anderson; Clarence A. Broomfield; Oksana Lockridge


Molecular Pharmacology | 1999

An Improved Cocaine Hydrolase: The A328Y Mutant of Human Butyrylcholinesterase is 4-fold More Efficient

Weihua Xie; Cibby Varkey Altamirano; Cynthia F. Bartels; Robert J. Speirs; John R. Cashman; Oksana Lockridge

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Patrick Masson

University of Wisconsin-Madison

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Lawrence M. Schopfer

University of Nebraska Medical Center

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Denis Josse

University of Nebraska Medical Center

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Cynthia F. Bartels

University of Nebraska Medical Center

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Angie Rizzino

Eppley Institute for Research in Cancer and Allied Diseases

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Judith A. Stribley

University of Nebraska Medical Center

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Phillip J. Wilder

Eppley Institute for Research in Cancer and Allied Diseases

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Steven H. Hinrichs

University of Nebraska Medical Center

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