Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency

Abstract

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.

characterization of the human monoacylglycerol lipase using mutagenesis, enzyme kinetics and NMR spectroscopy

“…Our previous NMR-based study succeeded in isolating the open and closed states of hMGL in solution and identified one of the key residues, His-272 responsible for the conformational stability [102]. We also confirmed that the flexible nature of hMGL enables global changes in the protein structure emerging from local dynamic changes [102].…”

“…The downfield region of the 1D NMR spectra (11-16ppm) of hMGL displays five distinct, well-separated and characteristic peaks. In our previous study, we definitively assigned four of these downfield peaks to hydrogen-bonded histidine residues based on a site-directed mutagenesis study that substituted histidines for alanine [102]. The downfield resonances at 15.9, 14.9, 13.9 and 12.8ppm in sol hMGL are therefore ascribed to residues His103, His269, His54 and His49 respectively; whereas the peak at 11 ppm belongs to a till-date unassigned -OH hydroxyl proton.…”

“…Recombinant sol-hMGL and the mutants in this study were expressed in E-coli BL21 cells and purified employing the IMAC technology as described previously [102].…”

“…MGL hydrolysis activity was measured chromatographically using the native substrate 2-AG [102]. The purified enzyme fractions were incubated for 20 min at 37 o C with increasing concentration range of 2-AG (13μM to 400μM).…”

“…The downfield resonances at 15.9, 14.9, 13.9 and 12.8ppm in sol hMGL are therefore ascribed to residues His103, His269, His54 and His49 respectively; whereas the peak at 11 ppm belongs to a till-date unassigned -OH hydroxyl proton. Moreover, we published how we developed an NMR-based method that exploits these histidine peaks as sensitive probes to study the conformational equilibrium and binding interactions [102]. Based on our previous studies, the closed conformation the interface of the core and lid domain has been extensively reviewed and confirmed to play a crucial role in catalysis [157].…”