Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843.
Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260.
Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):7617-7622. doi: 10.1073/pnas.1706134114. Epub 2017 Jul 5.
Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for () during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent -homopyruvoyl adduct of the active-site Cys.
异柠檬酸裂解酶（ICL，1 型和 2 型）是乙醛酸支路的第一个酶，该途径是人类结核病感染持续期（）所必需的。在这里，我们报道 2-乙烯基-d-异柠檬酸（2-VIC）是一种 1CL1 和 ICL2 的基于机制的失活剂。2-VIC 的酶促反醛醇裂解暴露出迈克尔底物 2-乙烯基甘油醛，然后它与活性位点半胱氨酸巯基形成缓慢可逆的共价加合物 2-VIC 显示出与 ICL1 和 ICL2 的共价、基于机制的高效失活（分区比，<1）一致的动力学特性。通过电喷雾电离质谱和 X 射线晶体学分析 ICL1：2-VIC 的复合物，证实了活性位点半胱氨酸的预测共价 -同型丙酮酸加合物的形成。
