Department of Chemistry, Center for Molecular Innovation and Drug Discovery, and Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois 60208, United States.
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, Illinois 60660, United States.
J Am Chem Soc. 2021 Jun 16;143(23):8689-8703. doi: 10.1021/jacs.1c02456. Epub 2021 Jun 7.
The inhibition of human ornithine δ-aminotransferase (OAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, ()-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, ) was identified as a potent mechanism-based inactivator of OAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of . A monofluorinated ketone () was identified as the primary metabolite of in OAT. By soaking OAT holoenzyme crystals with , a precursor to was successfully captured. This -diamine intermediate, covalently bound to Lys292, observed for the first time in OAT/ligand crystals, validates the turnover mechanism proposed for . Co-crystallization yielded OAT in complex with and revealed a novel noncovalent inactivation mechanism in OAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.
（-）-3-氨基-4,4-二氟环戊-1-烯羧酸（SS-1-148，）被鉴定为一种有效的人鸟氨酸 δ-氨基转移酶（OAT）抑制剂，对其他相关氨基转移酶（如 GABA-AT）具有优异的选择性。进行了综合的机制研究，以研究的周转和失活动力学。鉴定出单氟酮（）是 OAT 中 的主要代谢物。通过用 OAT 全酶晶体浸泡，成功捕获了的前体。首次在 OAT/配体晶体中观察到与 Lys292 共价结合的 -二胺中间体，验证了为 OAT 提出的周转机制。共结晶得到了与结合的 OAT，并揭示了 OAT 中的一种新型非共价失活机制。首次将天然蛋白质质谱用于研究氨基转移酶失活剂，以验证配体与酶之间的非共价相互作用；还鉴定了一种共价结合的复合物，作为在变性完整蛋白质质谱中观察到的次要形式。光谱和停流动力学实验支持赖氨酸辅助的 E2 氟离子消除，这在其他相关氨基转移酶失活剂的研究中从未观察到过。这种消除从初始外部醛亚胺直接产生第二个外部亚胺，而不是典型的 E1cB 消除机制，在两个外部亚胺之间形成醌型瞬变状态。天然蛋白质质谱、采用浸泡和共结晶方法的 X 射线晶体学以及停流动力学的使用允许详细阐明不寻常的周转和失活途径。
