Faculty of Biosciences, and Technology for Food Agriculture and Environment, University of Teramo, Teramo, Italy.
European Center for Brain Research (CERC)/Santa Lucia Foundation, Rome, Italy.
Sci Rep. 2020 Feb 10;10(1):2292. doi: 10.1038/s41598-020-59120-1.
Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.
脂肪酸酰胺水解酶（FAAH）是一种膜结合的同源二聚体酶，它在体内控制 N-花生四烯酰乙醇胺（AEA）和其他称为内源性大麻素的相关生物活性脂质的含量和生物活性。FAAH 单体的平行取向可能允许两个亚基同时募集和切割底物。在这里，我们通过以同源二聚体：抑制剂摩尔比为 1:1 的酶抑制剂完全抑制了人和大鼠 FAAH，这意味着仅占据 FAAH 的两个活性位点之一就足以完全阻断催化。大鼠 FAAH 中的单个 W445Y 取代显示出与野生型相同的活性，但在同源二聚体：抑制剂 1:1 比例下未能显示出完全抑制。相反，F432A 突变体表现出降低的比活性，但在同源二聚体：抑制剂 1:1 比例下完全被抑制。大鼠 FAAH 和其 F432A 突变体对 AEA 水解的动力学分析表明，Hill 系数约为 1.6，而 W445Y 突变体约为 1.0。值得注意的是，人 FAAH 也催化了 AEA 的别构水解，显示出约 1.9 的 Hill 系数。总之，这项研究证明了 FAAH 前所未有的变构作用，并且代表了在二聚体界面上似乎由单个氨基酸（W445）控制的两个酶亚基之间的通信的情况。鉴于在过去十年中为人类治疗开发有效药物的广泛尝试和随后的失败，这些发现为新分子的合理设计铺平了道路，这些新分子可以作为 FAAH 的正或负变构效应物，更有效地控制其活性。
