Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)
详细信息 查看全文
- 作者：Ioannis Karageorgos ; Nikolai Zvonok ; David R. Janero ; V. Kiran Vemuri ; Vidyanand Shukla ; Thomas E. Wales ; John R. Engen ; Alexandros Makriyannis
- 刊名：ACS Chemical Neuroscience
- 出版年：2012
- 出版时间：May 16, 2012
- 年：2012
- 卷：3
- 期：5
- 页码：393-399
- 全文大小：253K
- 年卷期：v.3,no.5(May 16, 2012)
- ISSN：1948-7193
文摘
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) desensitization 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) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. 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 and suggest that the nucleophilic character of the Cys122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. 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, the key role of the nucleophilic character of Ser122, and the mechanisms underlying hMGL inhibition by different classes of small molecules.