The Role of Active Site Hydrogen Bonding Network in the Enzymatic Reaction æCatalyzed by Monoacylglycerol Lipase æGirija Rajarshi, Ioannis Karageorgos, Sergiy Tyukhtenko, Nikolai Zvonok, and Alexandros Makriyannis æCenter for Drug Discovery, Northeastern University æHuman monoacylglycerol lipase (hMGL) is a serine hydrolase that regulates endocannabinoid signaling and is an attractive target for therapeutic intervention. To inhibit this enzyme potently and selectively, a detailed understanding of its structure and catalytic mechanism is essential. Here, we have used a æcombination of mutational, kinetic and NMR spectroscopy analyses to elucidate the catalytic role of hydrogen (H)-bonding network (HBN) and residues within the hMGL catalytic triad. Mutagenesis based æassignment of downfield 1H NMR resonance of His269 from the catalytic triad allows sensitive monitoring of active site structural rearrangements. Our data provides evidence that highly preorganized collection of normal hydrogen bonds within the hMGL catalytic triad is responsible for optimal positioning of all catalytically important residues. Modification or removal of any H-bonds involved in the catalytic triad preorganization severely compromised the HBN and significantly decreased enzyme turnover rate. Catalytic triad Ser122 or His269 replacements to Ala caused nearly total loss of activity, whereas Asp239 to Ala mutation retained some enzyme activity. æThus, biochemical profiling of hMGL mutants combined with NMR-based monitoring of perturbations in the active site may provide a useful tool to elucidate the functional roles of this enzymeÍs HBN and active site residues. æAcknowledgements: This work was supported by NIH grants DA003801 (A.M.), DA007215 (A.M.), and DA007312 (A.M.) from the NIDA.