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Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius

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Abstract

Kinetic studies of irreversible inhibition in recent years have received growing attention owing to their relevance to problems of basic scientific interest as well as to their practical importance. Our studies have been devoted to the characterization of the effects that well-known acetylcholinesterase irreversible inhibitors exert on a carboxylesterase (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius. In particular, sulfonyl inhibitors and the organophosphorous insecticide diethyl-p-nitrophenyl phosphate (paraoxon) have been studied. The incubation of EST2 with sulfonyl inhibitors resulted in a time-dependent inactivation according to a pseudo-first-order kinetics. On the other hand, the EST2 inactivation process elicited by paraoxon, being the inhibition reaction completed immediately after the inhibitor addition, cannot be described as a pseudo-first-order kinetics but is better considered as a high affinity inhibition. The values of apparent rate constants for paraoxon inactivation were determined by monitoring the enzyme/substrate reaction in the presence of the inhibitor, and were compared with those of the sulfonyl inhibitors. The protective effect afforded by a competitive inhibitor on the EST2 irreversible inhibition, and the reactivation of a complex enzyme/irreversible-inhibitor by hydroxylamine and 2-PAM, were also investigated. The data have been discussed in the light of the recently described dual substrate binding mode of EST2, considering that the irreversible inhibitors employed were able to discriminate between the two different binding sites.

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Abbreviations

EST2:

Esterase 2

HSL:

Hormone sensitive lipase

2-PAM:

2-(hydroxyiminomethyl)-1-methylpyridinium iodide

Paraoxon:

Diethyl-p-nitrophenyl phosphate

PMSF:

Phenylmethanesulfonyl fluoride

HDSC:

1-Hexadecanesulfonyl chloride

pNP-C6:

Nitrophenyl-hexanoate

pNP-C12:

Nitrophenyl-dodecanoate

HEPES:

2-[4-(2-hydroxyethyl)-1-piperazino]-ethansulfonic acid

References

  • Abou-Donia MB (2003) Organophosphorus ester-induced chronic neurotoxicity. Arch Environ Health 58:484–497

    Article  PubMed  CAS  Google Scholar 

  • Albero B, Sanchez-Brunete C, Tadeo JL (2003) Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography. J Agric Food Chem 51:6915–6921

    Article  PubMed  CAS  Google Scholar 

  • Bajgar J (2004) Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis, and treatment. Adv Clin Chem 38:151–216

    Article  PubMed  CAS  Google Scholar 

  • Barber DS, Ehrich M (2001) Esterase inhibition in SH-SY5Y human neuroblastoma cells following exposure to organophosphorus compounds for 28 days. In Vitr Mol Toxicol 14:129–135

    Article  PubMed  CAS  Google Scholar 

  • Bernabei M, Chiavarini S, Cremisini C, Palleschi G (1993) Anticholinesterase activity measurement by a choline biosensor: application in water analysis. Biosens Bioelectron 8:265–271

    Article  PubMed  CAS  Google Scholar 

  • Bhat JY, Shastri BG, Balaram H (2008) Kinetic and biochemical characterization of Plasmodium falciparum GMP synthetase. Biochem J 409:263–273

    Article  PubMed  CAS  Google Scholar 

  • Cao CJ, Mioduszewski RJ, Menking DE, Valdes JJ, Katz EJ, Eldefrawi ME, Eldefrawi AT (1999) Cytotoxicity of organophosphate anticholinesterases. In Vitro Cell Dev Biol Anim 35:493–500

    Article  PubMed  CAS  Google Scholar 

  • Carlson K, Jortner BS, Ehrich M (2000) Organophosphorus compound-induced apoptosis in SH-SY5Y human neuroblastoma cells. Toxicol Appl Pharmacol 168:102–113

    Article  PubMed  CAS  Google Scholar 

  • Cremisini C, Di Sario S, Mela J, Pilloton R, Palleschi G (1995) Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor. Anal Chim Acta 311:273–280

    Article  CAS  Google Scholar 

  • Demo SD, Kirk CJ, Aujay MA, Buchholz TJ, Dajee M, Ho MN, Jiang J, Laidig GJ, Lewis ER, Parlati F, Shenk KD, Smyth MS, Sun CM, Vallone MK, Woo TM, Molineaux CJ, Bennett MK (2007) Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res 67:6383–6391

    Article  PubMed  CAS  Google Scholar 

  • De Simone G, Manco G, Galdiero S, Lombardi A, Rossi M, Pavone V (1999) Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. Acta Crystallogr D Biol Crystallogr 55:1348–1349

    Article  PubMed  Google Scholar 

  • De Simone G, Galdiero S, Manco G, Lang D, Rossi M, Pedone C (2000) A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. J Mol Biol 303:761–771

    Article  PubMed  CAS  Google Scholar 

  • De Simone G, Menchise V, Alterio V, Mandrich L, Rossi M, Manco G, Pedone C (2004a) The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family. J Mol Biol 343:137–146

    Article  PubMed  CAS  Google Scholar 

  • De Simone G, Mandrich L, Menchise V, Giordano V, Febbraio F, Rossi M, Pedone C, Manco G (2004b) A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis. J Biol Chem 279:6815–6823

    Article  PubMed  CAS  Google Scholar 

  • Dixon M, Webb EC (1979) Enzymes, 3rd edn. Academic Press, New York

    Google Scholar 

  • Eckert S, Eyer P, MÜckter H, Worek F (2006) Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds. Biochem Pharmacol 72:344–357

    Article  PubMed  CAS  Google Scholar 

  • Febbraio F, Barone R, D’Auria S, Rossi M, Nucci R, Piccialli G, De Napoli L, Orru S, Pucci P (1997) Identification of the active site nucleophile in the thermostable β-glycosidase from the archaeon Sulfolobus solfataricus expressed in Escherichia coli. Biochemistry 36:3068–3075

    Article  PubMed  CAS  Google Scholar 

  • Forsberg A, Puu G (1984) Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates. Eur J Biochem 140:153–156

    Article  PubMed  CAS  Google Scholar 

  • Hemingway J, Karunaratne SH (1998) Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Med Vet Entomol 12:1–12

    Article  PubMed  CAS  Google Scholar 

  • Hugonnet JE, Blanchard JS (2007) Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate. Biochemistry 46:11998–12004

    Article  PubMed  CAS  Google Scholar 

  • Karunaratne SH, Jayawardena KG, Hemingway J, Ketterman AJ (1993) Characterization of a B-type esterase involved in insecticide resistance from the mosquito Culex quinquefasciatus. Biochem J 294:575–579

    PubMed  CAS  Google Scholar 

  • Legler G, Harder A (1978) Amino acid sequence at the active site of β-glucosidase A from bitter almonds. Biochim Biophys Acta 524:102–108

    PubMed  CAS  Google Scholar 

  • Leytus SP, Toledo DL, Mangel WF (1984) Theory and experimental method for determining individual kinetic constants of fast-acting, irreversible proteinase inhibitors. Biochim Biophys Acta 788:74–86

    PubMed  CAS  Google Scholar 

  • Liu W, Tsou CL (1986) Determination of rate constants for the irreversible inhibition of acetylcholine esterase by continuously monitoring the substrate reaction in the presence of the inhibitor. Biochim Biophys Acta 870:185–190

    PubMed  CAS  Google Scholar 

  • Lu J, Chew EH, Holmgren A (2007) Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide. Proc Natl Acad Sci USA 104:12288–12293

    Article  PubMed  CAS  Google Scholar 

  • Maklakov A, Ishaaya I, Freidberg A, Yawetz A, Horowitz AR, Yarom I (2001) Toxicological studies of organophosphate and pyrethroid insecticides for controlling the fruit fly Dacus ciliatus (Diptera: Tephritidae). J Econ Entomol 94:1059–1066

    Article  PubMed  CAS  Google Scholar 

  • Manco G, Adinolfi E, Pisani FM, Carratore V, Rossi M (1997) Identification of an esterase from Bacillus acidocaldarius with sequence similarity to a hormone sensitive lipase subfamily. Pept Lett 4:375–382

    CAS  Google Scholar 

  • Manco G, Adinolfi E, Pisani FM, Ottolina G, Carrea G, Rossi M (1998) Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone sensitive lipase subfamily. Biochem J 332:203–212

    PubMed  CAS  Google Scholar 

  • Manco G, Febbraio F, Adinolfi E, Rossi M (1999) Homology modeling and active site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2. Prot Sci 8:1789–1796

    Article  CAS  Google Scholar 

  • Manco G, Mandrich L, Rossi M (2001) Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature. J Biol Chem 276:37482–37490

    Article  PubMed  CAS  Google Scholar 

  • Manco G, Carrea G, Giosue E, Ottolina G, Adamo G, Rossi M (2002) Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening. Extremophiles 6:325–331

    Article  PubMed  CAS  Google Scholar 

  • Mionetto N, Marty JL, Karube I (1994) Acetylcholinesterase in organic solvents for the detection of pesticides: biosensor application. Biosens Bioelectron 9:463–470

    Article  CAS  Google Scholar 

  • Naravaneni R, Jamil K (2007) Determination of AChE levels and genotoxic effects in farmers occupationally exposed to pesticides. Hum Exp Toxicol 26:723–731

    Article  PubMed  CAS  Google Scholar 

  • Pyatakova NV, Grigoryev NB, Severina IS (1999) Role of soluble guanylate cyclase in reactivation of choline esterase inhibited by phosphoorganic compounds. Biochemistry (Mosc) 64:91–94

    CAS  Google Scholar 

  • Saleh AM, Vijayasarathy C, Fernandez-Cabezudo M, Taleb M, Petroianu G (2003) Influence of paraoxon (POX) and parathion (PAT) on apoptosis: a possible mechanism for toxicity in low-dose exposure. J Appl Toxicol 23:23–29

    Article  PubMed  CAS  Google Scholar 

  • Small GJ, Karunaratne SH, Chadee DD, Hemingway J (1999) Molecular and kinetic evidence for allelic variants of esterase Estβ1 in the mosquito Culex quinquefasciatus. Med Vet Entomol 13:274–281

    Article  PubMed  CAS  Google Scholar 

  • Sun X, Liu XB, Martinez JR, Zhang GH (2000) Effects of low concentrations of paraoxon on Ca(2+) mobilization in a human parotid salivary cell-line HSY. Arch Oral Biol 45:621–638

    Article  PubMed  CAS  Google Scholar 

  • Tian WX, Tsou CL (1982) Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier. Biochemistry 21:1028–1032

    Article  PubMed  CAS  Google Scholar 

  • Tull D, Withers SG, Gilkes NR, Kilburn DG, Warren RA, Aebersold R (1991) Glutamic acid 274 is the nucleophile in the active site of a “retaining” exoglucanase from Cellulomonas fimi. J Biol Chem 266:15621–15625

    PubMed  CAS  Google Scholar 

  • Vilanova E, Vicedo JL (1983) Serum cholinesterase inhibitors in the commercial hexane impurities. Arch Toxicol 53:59–69

    Article  PubMed  CAS  Google Scholar 

  • Withers SG, Aebersold R (1995) Approaches to labeling and identification of active site residues in glycosidases. Prot Sci 4:361–372

    Article  CAS  Google Scholar 

  • Worek F, Kirchner T, Backer M, Szinicz L (1996) Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphorus compounds. Arch Toxicol 70:497–503

    Article  PubMed  CAS  Google Scholar 

  • Zdrazilová P, Stĕpánková S, Komersová A, Vránová M, Komers K, Cegan A (2006) Kinetics of 13 new cholinesterase inhibitors. Z Naturforsch [C] 61:611–617

    Google Scholar 

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Authors and Affiliations

  1. Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy

    Ferdinando Febbraio, Sandro Esposito D’Andrea, Luigi Mandrich, Luigia Merone, Mosè Rossi, Roberto Nucci & Giuseppe Manco

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  1. Ferdinando Febbraio
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  2. Sandro Esposito D’Andrea
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  3. Luigi Mandrich
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  4. Luigia Merone
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  5. Mosè Rossi
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  6. Roberto Nucci
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  7. Giuseppe Manco
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Corresponding author

Correspondence to Ferdinando Febbraio.

Additional information

Communicated by A. Driessen.

This work was supported by a grant to G. M. from “Regione Campania” Year 2000.

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Febbraio, F., D’Andrea, S.E., Mandrich, L. et al. Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius . Extremophiles 12, 719–728 (2008). https://doi.org/10.1007/s00792-008-0179-1

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  • DOI: https://doi.org/10.1007/s00792-008-0179-1

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