title: Mutation of Cys242 of human monoacylglycerol lipase disrupts balanced hydrolysis of 1- and 2-monoacylglycerols and selectively impairs inhibitor potency.
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.
authors.affiliation: 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.
authors.forename: Tuomo
authors.initials: T
authors.lastname: Laitinen
authors.forename: Dina
authors.initials: D
authors.lastname: Navia-Paldanius
authors.forename: Roosa
authors.initials: R
authors.lastname: Rytilahti
authors.forename: Joona J T
authors.initials: JJ
authors.lastname: Marjamaa
authors.forename: Julie
authors.initials: J
authors.lastname: Kařízková
authors.forename: Teija
authors.initials: T
authors.lastname: Parkkari
authors.forename: Tatu
authors.initials: T
authors.lastname: Pantsar
authors.forename: Antti
authors.initials: A
authors.lastname: Poso
authors.forename: Jarmo T
authors.initials: JT
authors.lastname: Laitinen
authors.forename: Juha R
authors.initials: JR
authors.lastname: Savinainen
mesh_terms: D001095:Arachidonic Acids / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D063388:Endocannabinoids / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005989:Glycerides / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D008994:Monoacylglycerol Lipases / Q000235:genetics* / Q000378:metabolism; D050178:Monoglycerides / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D050178:Monoglycerides; C094503:glyceryl 2-arachidonate; D008994:Monoacylglycerol Lipases; D003545:Cysteine