Isocitrate lyase (ICL) from Mycobacterium tuberculosis is a key enzyme of the glyoxylate shunt required for survival during latent infection and is absent in humans. Maleate has long been characterized as a reversible competitive inhibitor that mimics the succinate product of catalysis. Here, we show that maleate instead functions as a slow, time-dependent covalent inactivator of ICL. Kinetic analysis supports a two-step mechanism involving reversible binding followed by irreversible modification of the catalytic cysteine residue, Cys191. Mass spectrometric analysis confirms covalent modification and, in the presence of glyoxylate, reveals an additional adduct incorporating both maleate and glyoxylate. Formation of this higher-mass species is consistent with enolate-like reactivity within the active site. Comparison with structurally related maleate analogues demonstrates that minimal substitution at the alkene abolishes covalent reactivity and alters the binding mode, highlighting the stringent geometric and electronic constraints imposed by the succinate-binding pocket. Together, these findings redefine the interaction of succinate analogues with ICL and provide mechanistic insight into active-site organization.
Fernando et al. (Thu,) studied this question.