title: Identification by nuclear magnetic resonance spectroscopy of an active-site hydrogen-bond network in human monoacylglycerol lipase (hMGL): implications for hMGL dynamics, pharmacological inhibition, and catalytic mechanism.
abstract: Intramolecular hydrogen bonding is an important determinant of enzyme structure, catalysis, and inhibitor action. Monoacylglycerol lipase (MGL) modulates cannabinergic signaling as the main enzyme responsible for deactivating 2-arachidonoylglycerol (2-AG), a primary endocannabinoid lipid messenger. By enhancing tissue-protective 2-AG tone, targeted MGL inhibitors hold therapeutic promise for managing pain and treating inflammatory and neurodegenerative diseases. We report study of purified, solubilized human MGL (hMGL) to explore the details of hMGL catalysis by using two known covalent hMGL inhibitors, the carbamoyl tetrazole AM6701 and N-arachidonoylmaleimide (NAM), that act through distinct mechanisms. Using proton nuclear magnetic resonance spectroscopy (NMR) with purified wild-type and mutant hMGLs, we have directly observed a strong hydrogen-bond network involving Asp239 and His269 of the catalytic triad and neighboring Leu241 and Cys242 residues. hMGL inhibition by AM6701 alters this hydrogen-bonding pattern through subtle active-site structural rearrangements without influencing hydrogen-bond occupancies. Rapid carbamoylation of hMGL Ser122 by AM6701 and elimination of the leaving group is followed by a slow hydrolysis of the carbamate group, ultimately regenerating catalytically competent hMGL. In contrast, hMGL titration with NAM, which leads to cysteine alkylation, stoichiometrically decreases the population of the active-site hydrogen bonds. NAM prevents reformation of this network, and in this manner inhibits hMGL irreversibly. These data provide detailed molecular insight into the distinctive mechanisms of two covalent hMGL inhibitors and implicate a hydrogen-bond network as a structural feature of hMGL catalytic function.
authors.affiliation: Center for Drug Discovery, Northeastern University, 116 Mugar Hall, Boston, MA 02115-5000, USA.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Karageorgos
authors.forename: Sergiy
authors.initials: S
authors.lastname: Tyukhtenko
authors.forename: Nikolai
authors.initials: N
authors.lastname: Zvonok
authors.forename: David R
authors.initials: DR
authors.lastname: Janero
authors.forename: Christine
authors.initials: C
authors.lastname: Sallum
authors.forename: Alexandros
authors.initials: A
authors.lastname: Makriyannis
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics*; D006801:Humans; D006860:Hydrogen Bonding / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008994:Monoacylglycerol Lipases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013777:Tetrazoles / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: C553459:AM 6701; D004791:Enzyme Inhibitors; D013777:Tetrazoles; D008994:Monoacylglycerol Lipases
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