Mechanism-based inactivator of isocitrate lyases 1 and 2 from .

Proceedings of the National Academy of Sciences of the United States of America, Volume: 114, Issue: 29
July 18, 2017
Truc V Pham TV, Andrew S Murkin AS, Margaret M Moynihan MM, Lawrence Harris L, Peter C Tyler PC, Nishant Shetty N, James C Sacchettini JC, Hsiao-Ling Huang HL, Thomas D Meek TD

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.

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