Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation

Download Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation

Post on 05-Sep-2016

215 views

Category:

Documents

2 download

TRANSCRIPT

  • Chemico-Biological Interactions 187 (2010) 238240

    Contents lists available at ScienceDirect

    Chemico-Biological Interactions

    journa l homepage: www.e lsev ier .com/ l

    Investigating the structural inuence of surface macetylcholinesterase inhibition by organophosphand oxime reactivation

    Tuba Kc dznGabi Ama Skaggs Schoo la, CAb Hacettepe Un keyc Department o te, La Jd Division of M 0, Isra

    a r t i c l

    Article history:Available online 9 April 2010

    Keywords:AcetylcholinesteraseOrganophosphatesOxime reactivCatalysisSurface mutat

    Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and toa lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of vary-ing structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. Inthis study wild type human AChE and human AChE with residue mutations D134H, D134H E202Q andD134H F338A were characterized and investigated for inhibition by OPs and consequent oxime reacti-

    1. Introdu

    To minimbinary mixtturewere rethe catalysiin skeletal msite-specicvert humanan oxime, wof scavenge

    Abbreviatiohuman AChE;wt, wild type;

    Corresponment of Biochfax: +90 312 3

    E-mail add(T. Kckkilin

    0009-2797/$ doi:10.1016/j.ation

    ions

    vation of phosphylated enzymes. The rationale for selecting these substitution positions was based onD134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q andF338A mutations slow aging of OP inhibited AChEs.

    Inhibition of D134H by paraoxon and analogues of cyclosarin was 28 times slower than inhibition ofwild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Bothinhibition and reactivation of D134H E202Q and D134H F338A double mutants were up to two orders ofmagnitude slower than the wt indicating that introduction of the active center substitutions abolishedfully the effect of the peripherally located D134H. These results indicate that selected residues outsidethe active center inuence inhibition, reactivation and catalysis rates through longer range interactions.

    2010 Elsevier Ireland Ltd. All rights reserved.

    ction

    ize the toxicity of organophosphate (OP) inhibitors,ures of mammalian AChE and oximes of varying struc-cently consideredas catalytic bioscavengerspromotings of the OP in plasma before it reacts with the target siteuscle or the nervous system [1]. Our goal is to generatemutations in AChE protein sequence in order to con-AChE into catalytic scavenger that, when coupled withill inactivate multiple OP molecules per one moleculer.

    ns: OPs, organophosphates; AChE, acetylcholinesterase; hAChE,BChE, butyrylcholinesterase; SNP, single nucleotide polymorphism;ATCh, acetylthiocholine; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid).ding author at: Hacettepe University, Faculty of Pharmacy, Depart-emistry, Sihhiye 06100, Ankara, Turkey. Tel.: +90 312 3051499;114777.resses: ttuylu@hacettepe.edu.tr, tubatuylu@gmail.comc).

    Human AChE wild type and D132H, D134H E202Q, andD134H F338A mutants were characterized and investigated forinhibition by OPs (an insecticide and nerve agent analogues; Fig. 1)and reactivation of the phosphylated enzymes with 2PAM and HI6.The rationale for selecting these substitution positions was basedon D134H being a naturally occurring SNP in humans and thatE202Qand F338Amutations slowaging ofOP inhibitedAChEs [2,3].

    2. Materials and methods

    Nerve agent analogues of cyclosarin (methylphosphonicacid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester cyclohexylester), VX (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester ethyl ester), soman (methylphosphonicacid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester pinacolylester) were synthesized as previously described [4], as well asSP-Cycloheptyl methyl phosphonyl thiocholine (SPCHMP) and RP-Isopropylmethylphosphonyl thiocholine (RPIPMP) [5].O,O-DiethylO-(4-nitrophenyl) phosphate (paraoxon) and the oxime 2PAMwere purchased from SigmaAldrich (St. Louis, MO). The oxime

    see front matter 2010 Elsevier Ireland Ltd. All rights reserved.cbi.2010.03.050 kkilinc a,b,, Rory Cochrana, Jaroslaw Kalisiakc, Eitaid, Zoran Radic a, Palmer Taylora

    l of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Joliversity, Faculty of Pharmacy, Department of Biochemistry, Sihhiye 06100, Ankara, Turf Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Instituedicinal Chemistry, Israel Institute for Biological Research, P.O. Box 19, Ness Ziona 7410

    e i n f o a b s t r a c tocate /chembio int

    utations onorus compounds

    a Garciaa, Anne Vallea,

    92093-0650, USA

    olla, CA 92037, USAel

  • T. Kckkilinc et al. / Chemico-Biological Interactions 187 (2010) 238240 239

    Fig. 1. Structures of OP inhibitors used in this study.

    HI6 was purchased from US Biological (Swampscott, MA). Oximeswereused in a rangeof 0.0510mM.Final concentrationsof organicsolvents were less than 1% in enzyme assays.

    HEK-293 cells were transfected with the hAChE D134H DNAconstruct, prepared as described for the mouse AChE [6], withexception that hAChE constructs had FLAG peptide sequenceencoded at the 5 end of the construct. The stable cell clones wereselected by treatment with G-418. The protein was puried in mgquantities by adsorption and desorption from an antiFLAG peptideantibody resin.

    In reactivation experiments wild type and mutant enzymes(40nM) were inhibited for 1030min until inhibition was greaterthan 95%. Inhibited enzyme and the control were passed throughtwo consecutive Sephadex G-50 spin columns to remove excessunreacted inhibitor. Inhibited and control enzymeswere incubatedwith oximemeasured itures by spphosphateand 333Mof experim[6]. Inhibitibefore [7]50M.

    Table 1Relative inhibition rates of hD134H wt; hD134H E202Q and hD134H F338A.

    Enzyme Paraoxon VX analogue Cyclosarinanalogue

    Somananalogue

    hAChE wta 1 1 1 1hD134H 0.4 1 0.1 1hD134H E202Q 0.5 0.2 0.01 0.04hD134H F338A 0.1 1 0.2 0.3

    The second-order inhibition rate constants of hAChE wt with paraoxon and ana-logues of VX, cyclosarin and soman were 330104, 290104, 580104 and60104 M1 min1, respectively. Inhibition by SPCHMPTCh, RPIPMPTCh was notmeasured.

    3. Results and discussion

    ibition of human D134H with paraoxon and an analogue ofrin was 28 times slower than that of human wt (Table 1),

    as inh4H subletude.M reer hdy i

    Table 2Relative reacti

    hAChE E Relative reactivation rate

    wta 1D134H 6D134H E202 0.1D134H F338 1wtb 1D134H 3

    wtc 1D134H 2wtd 1D134H 0.3D134H E202 0.02D134H F338 0.002

    wte

    D134H

    wtf

    D134H

    The second-ora 120M1 mb 45M1 mic 275M1 md 13,000Me 490M1 mf 270M1 mand time courses of recovery of hAChE activity weren aliquots of reactivation and control reaction mix-ectrophotometric Ellman assay (at 22 C, in 100mMbuffer, pH 7.4, containing 1mM ATCh as substrate

    DTNB as nal concentrations) Detailed descriptionental procedures and calculations is given elsewhereon kinetic experiments were conducted as describedusing nal OP concentrations between 10nM and

    Inhcyclosawhereby D13two domagni

    2PAthe othous stu

    vation rates of OP inhibited hD134H; hD134H E202Q and hD134H F338A.

    OP OXIM

    Paraoxon 2PAM

    QA

    HI6

    SPCHMPTCh 2PAM

    HI6

    QAVX analogue 2PAM

    RPIPMPTCh 2PAM

    der rate constants for reactivation of OP inhibited wt hAChE by oximes were as follows.in1 (paraoxon inhibited wt hAChE reactivated by 2PAM).n1 (paraoxon inhibited wt hAChE reactivated by HI6).in1 (SPCHMPTCh inhibited wt hAChE reactivated by 2PAM).1 min1 (SPCHMPTCh inhibited wt hAChE reactivated by HI6).in1 (VX inhibited wt hAChE reactivated by 2PAM).in1 (RPIPMPTCh inhibited wt hAChE reactivated by 2PAM). Reactivation of cyclosarin anibition by soman and analogues of VX was not affectedubstitution. Introduction of additional substitutions inmutants further slowed inhibition up to two orders of

    activation of paraoxon inhibited human D134H is, onand, 6 times faster than human wt (Table 2). Our previ-ndicated that differential occupation of the acyl pocket11

    11

    d soman inhibited hAChE was not measured.

  • 240 T. Kckkilinc et al. / Chemico-Biological Interactions 187 (2010) 238240

    in mouse AChE, by covalent ligands of varying geometry, had dis-tinct effects on spectra of uorophores covalently attached on theAChE surface in the general spatial vicinity of residue D134 [8].In order to determine whether the acyl pocket stabilized ethoxysubstituent on phosphorus in diethylphosphorylated hAChE wasprimarily responsible for the observed enhancement of reactiva-tion rates, the enzymes were inhibited with an excess of racemicVX analogue. Inhibition was assumed to yield wt and D134H AChEsconjugated primarily by the SP VX enantiomer with methylphos-phonyl substituent of the conjugate stabilized in the acyl pocketand the ethoxy substituent oriented towards the choline bindingsite. Reactivation of both wt and D134H hAChE SP VX conjugateswith2PAMshowed similar reactivation rates. This suggests that the2PAM reactivation rate enhancement observed in the diethylphos-phorylated (paraoxon inhibited)D134Hmutantmaybe linkedwithimproved stabilization of the ethoxy substituent in the mutant acylpocket and is consistentwith theobserved three-fold enhancementof the HI6 reactivation rate (Table 2). To verify this hypothesis fur-ther 2PAM reactivation of the RPIPMP inhibited enzyme conjugates(identical to RP Sarin conjugated AChE) was studied but yielded noeffect of D134H mutation. Thus, our experiments implicate differ-ential exibility of the acyl pocket in D134H substituted hAChE,but the actual mechanism of the 2PAM reactivation enhancementremains unclear.

    Human D134H E202Q and D134H F338A non-aging doublemutants did not reactivate any faster than the wt or the hD134Hmutant (Table 2). In fact, of all tested enzymes the one most com-promised with mutations was D134H E202Q mutant. It showedboth inhibition and reactivation rates 20 times slower in compari-son to wt and D134H (Tables 1 and 2). The characteristics of doublemutantsmostly arose fromE202Qmutation thathas slowphospho-nylation anresistance tand enhancthe same do

    In summresidue subthe active coxime reac

    couples for catalytic OP hydrolysis. However, combining diverseproperties of single site mutations to form multiple site hAChEmutants does not result in a simple summation of G values, ratherthere is a coupling of energy values that appear linked.

    Conict of interest statement

    None declared.

    Acknowledgements

    This study was supported by fellowships from IIE and The Ful-bright Program, and the Scientic and Technical Research Councilof Turkey to Tuba Kucukkilinc and by NIH NINDS U01NS58046 toPalmer Taylor.

    References

    [1] P. Taylor, Z. Kovarik, E. Reiner, Z. Radic, Acetylcholinesterase: converting a vul-nerable target to a template for antidotes and detection of inhibitor exposure,Toxicology 233 (2007) 7078.

    [2] A. Saxena, B.P. Doctor, D.M. Maxwell, D.E. Lenz, Z. Radic, P. Taylor, The role ofglutamate-199 in the aging of cholinesterase, Biochem. Biophys. Res. Commun.197 (1993) 343349.

    [3] A. Shafferman, A. Ordentlich, D. Barak, D. Stein, N. Ariel, B. Velan, Aging ofphosphylated human acetylcholinesterase: catalytic processes mediated byaromatic and polar residues of the active centre, Biochem. J. 318 (1996)833840.

    [4] G. Amitai, R. Adani, G. Yacov, S. Yishay, S. Teitlboim, L. Tveria, O. Limanovich, M.Kushnir, H.Meshulam,Asymmetric uorogenic organophosphates for thedevel-opment of active organophosphate hydrolases with reversed stereoselectivity,Toxicology 233 (2007) 187198.

    [5] H.A. Berman, K. Leonard, Chiral reactions of acetylcholinesterase probed withenantiomeric methylphosphonothioates. Noncovalent determinants of enzymechirality, J. Biol. Chem. 264 (1989) 39423950.

    varik,inesteted coovarikylcholtorial0., A.E. Binduc

    ylchold reactivation rates. We were thus unable to combineo aging (coming from E202Q or F338A substitutions)ed reactivation (coming from D134H substitution) inuble mutant hAChE protein.ary, our initial study shows that selected surface

    stitutions in the AChE molecule located remotely fromenter affect its catalytic parameters, OP inhibition andtivation and may be useful in designing oxime-hAChE

    [6] Z. Kocholnyla

    [7] Z. KAcetbina334

    [8] J. ShiandsacetZ. Radic, H.A. Berman, V. Simeon-Rudolf, E. Reiner, P. Taylor, Mutantrases possessing enhanced capacity for reactivation of their phospho-njugates, Biochemistry 43 (2004) 32223229., Z. Radic, H.A. Berman, V. Simeon-Rudolf, E. Reiner, P. Taylor,inesterase active centre and gorge conformations analysed by com-mutations and enantiomeric phosphonates, Biochem. J. 373 (2003)

    oyd, Z. Radic, P. Taylor, Reversibly bound and covalently attached lig-e conformational changes in the omega loop, Cys69-Cys96, of mouseinesterase, J. Biol. Chem. 276 (2001) 4219642204.

    Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compoun...IntroductionMaterials and methodsResults and discussionConflict of interest statementAcknowledgementsReferences

Recommended

View more >