Abstract β-Bromopyruvate (BrP) alkylates isocitrate lyase at the active site as indicated by the following: (1) the irreversible alkylation shows saturation kinetics, (2) substrate, isocitrate, is a competitive inhibitor of inactivation by BrP, (3) the combination of products, glyoxylate and succinate, or a competitive inhibitor of the enzyme, oxalate, or glyoxylate plus itaconate (a succinate analogue) all protect against inactivation by BrP, (4) the rate of inactivation can be increased by EDTA, an initiator of the catalytic reaction in the presence of activated (reduced) enzyme and substrate. About four carboxyketomethyl residues are incorporated per mole of inactivated enzyme which implies four catalytic sites in the tetrameric enzyme. Amino acid analysis after H2O2 treatment of the alkylated enzyme demonstrates that one cys/monomer is being alkylated.