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Molecular Pharmacology

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Research ArticleArticle

Mutation of Cys242 of Human Monoacylglycerol Lipase Disrupts Balanced Hydrolysis of 1- and 2-Monoacylglycerols and Selectively Impairs Inhibitor Potency

Tuomo Laitinen, Dina Navia-Paldanius, Roosa Rytilahti, Joona J. T. Marjamaa, Julie Kařízková, Teija Parkkari, Tatu Pantsar, Antti Poso, Jarmo T. Laitinen and Juha R. Savinainen
Molecular Pharmacology March 2014, 85 (3) 510-519; DOI: https://doi.org/10.1124/mol.113.090795
Tuomo Laitinen
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Dina Navia-Paldanius
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Roosa Rytilahti
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Joona J. T. Marjamaa
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Julie Kařízková
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Teija Parkkari
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Tatu Pantsar
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Antti Poso
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Jarmo T. Laitinen
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Juha R. Savinainen
Institute of Biomedicine/Physiology, School of Medicine (D.N.-P., R.R., J.J.T.M., J.K., Te.P., J.T.L., J.R.S.), and School of Pharmacy (T.L., Ta.P., A.P.), Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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Abstract

Considerable progress has been made in recent years in developing selective, potent monoacylglycerol lipase (MAGL) inhibitors. In the investigations of measures to inhibit this enzyme, less attention has been paid to improving our understanding of its catalytic mechanisms or substrate preferences. In our study, we used site-directed mutagenesis, and we show via versatile activity assays combined with molecular modeling that Cys242 and Tyr194, the two opposing amino acid residues in the catalytic cavity of MAGL, play important roles in determining the rate and the isomer preferences of monoacylglycerol hydrolysis. In contrast to wild-type enzymes that hydrolyzed 1- and 2-monoacylglycerols at similar rates, mutation of Cys242 to alanine caused a significant reduction in overall activity (maximal velocity, Vmax), particularly skewing the balanced hydrolysis of isomers to favor the 2-isomer. Molecular modeling studies indicate that this was caused by structural features unfavorable toward 1-isomers as well as impaired recognition of OH-groups in the glycerol moiety. Direct functional involvement of Cys242 in the catalysis was found unlikely due to the remote distance from the catalytic serine. Unlike C242A, mutation of Tyr194 did not bias the hydrolysis of 1- and 2-monoacylglycerols but significantly compromised overall activity. Finally, mutation of Cys242 was also found to impair inhibition of MAGL, especially that by fluorophosphonate derivatives (13- to 63-fold reduction in potency). Taken together, this study provides new experimental and modeling insights into the molecular mechanisms of MAGL-catalyzed hydrolysis of the primary endocannabinoid 2-arachidonoylglycerol and related monoacylglycerols.

Footnotes

    • Received November 15, 2013.
    • Accepted December 24, 2013.
  • This work was supported by Biocenter Finland/DDCB (to T.L.); the Finnish Cultural Foundation (N-S Regional Fund) and Kuopio University Foundation (to J.R.S.); and the Academy of Finland [Grant 139620] (to J.T.L.).

  • J.K., a student of pharmacy from Prague, Czech Republic, participated in this study as an ERASMUS exchange student.

  • dx.doi.org/10.1124/mol.113.090795.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 85 (3)
Molecular Pharmacology
Vol. 85, Issue 3
1 Mar 2014
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Research ArticleArticle

Structural Determinants of MAGL for Substrate Preference

Tuomo Laitinen, Dina Navia-Paldanius, Roosa Rytilahti, Joona J. T. Marjamaa, Julie Kařízková, Teija Parkkari, Tatu Pantsar, Antti Poso, Jarmo T. Laitinen and Juha R. Savinainen
Molecular Pharmacology March 1, 2014, 85 (3) 510-519; DOI: https://doi.org/10.1124/mol.113.090795

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Research ArticleArticle

Structural Determinants of MAGL for Substrate Preference

Tuomo Laitinen, Dina Navia-Paldanius, Roosa Rytilahti, Joona J. T. Marjamaa, Julie Kařízková, Teija Parkkari, Tatu Pantsar, Antti Poso, Jarmo T. Laitinen and Juha R. Savinainen
Molecular Pharmacology March 1, 2014, 85 (3) 510-519; DOI: https://doi.org/10.1124/mol.113.090795
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