Considerable progress has been made in recent years in developing selective and potent monoacylglycerol lipase (MAGL) inhibitors. In pursuance of investigating measures to inhibit this enzyme, less attention has been paid to improve our understanding on its catalytic mechanisms or substrate preference. In this study, we utilized site-directed mutagenesis and show by using 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 isomer preference of monoacylglycerol hydrolysis. In contrast to wild type enzyme, which hydrolyzes 1- and 2-monoacylglycerols with similar rates, mutation of Cys242 to alanine caused a significant reduction in overall activity (maximal velocity, Vmax) and particularly, skewed the balanced hydrolysis of isomers to favor the 2-isomer. Molecular modeling studies indicated that this was caused by unfavorable structural features towards 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 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 (13-63-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.

PMID:

 

24368842

 

[PubMed – as supplied by publisher]