ATP-citrate lyase (ACLY) synthesizes acetyl-CoA and oxaloacetate from citrate, CoA and ATP, with citryl-CoA as an intermediate. Current insight into the mechanism of cleavage of citryl-CoA is based on sequence similarity of ACLY to citrate synthase (CS). Histidine and aspartic acid that are active site residues in CS are conserved in ACLY. Based on the hypothesis that these two amino acid residues are needed for catalytic activity, these residues were mutated to alanine in ACLY from Chlorobium limicola (bACLY). Enzymatic assays of the mutants H491αA showed 64% decrease in specific activity compared to wild-type; while D543αA and double-mutant (H491αA, D543αA) were not active. Biophysical analyses, including kinetics assays, isothermal titration calorimetry and differential scanning fluoriometry, were done to determine whether the mutated residues altered the enzyme’s structural conformation. Results from biophysical analyses suggest that the reduction in specific activity was due to the loss of side-chain and not a conformational change. Therefore, H491αA and D543αA in bACLY are involved in maintaining maximum catalytic activity.