Endocannabinoid Enzyme Engineering: Soluble Human Thio-monoacylglycerol Lipase (sol-S-hMGL)

Abstract

Endocannabinoid Enzyme Engineering: Soluble Human Thio-monoacylglycerol Lipase (sol-S-hMGL) In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) sensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu169;Leu176)-to-Ser(Ser169;Ser176) double hMGL mutant (sol-hMGL) and further mutated this variant by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorescent reporter substrate with comparable affinities, but the Ser122-to-Cys substitution markedly slowed the rate of hydrolysis. The interaction of each hMGL variant with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser122 residue as compared to that of Cys122 in the sol-S-hMGL mutant. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis and the mechanisms underlying hMGL inhibition by different classes of small molecules. Supported by grants from NIH 3801 and 9158