a, The four steps in the reaction catalysed by ACLY. b, Production of ADP is linear over 60 min. Data from duplicate experiments are shown, but the linearity of the progress curve was observed in many additional experiments. c–e, Double reciprocal plots showing that NDI-091143 is competitive versus citrate (Ki = 7.0 ± 0.8 nM) (c); noncompetitive versus ATP (Ki = 218 ± 14 nM) (d); and mixed-type noncompetitive versus CoA (Ki = 5.0 ± 2.1 nM, Ki′ = 156 ± 20 nM) (e). Data from duplicate experiments are shown (n = 2). Although the error on the Ki value versus CoA is relatively high, it does not affect the conclusion regarding the pattern of inhibition versus CoA. The assigned pattern of inhibition has 99.99% probability based on global fitting. The fixed substrate concentrations were 300 μM ATP, 300 μM CoA, and 5 mM citrate. f, Limited proteolysis experiments on full-length human ACLY wild-type (WT), H760A, and H760E mutants in the presence of various substrates and/or NDI-091143. CHY, chymotrypsin. The experiments were repeated at least three times, with similar results.