Coenzyme A (CoA) is found in all living organisms and is involved in numerous cellular biochemical reactions. CoA and its derivatives function as cofactors in many cellular pathways and are involved in protein regulation through post-translational modifications as well as competitive and allosteric interactions. Deregulation of CoA has implications in various human diseases including cancer, diabetes and neurodegeneration. Given the importance of CoA, I synthesized a biotin-tagged CoA probe to identify CoA-binding proteins. Interestingly, my chemical proteomic approach identified several metabolic enzymes that are not known to utilize CoA. I biochemically validated the top hit, mitochondrial malic enzyme 2 (ME2), as a CoA-binding protein. ME2 is a NAD(P)-dependent enzyme that converts malate to pyruvate and at the same time generates NAD(P)H. ME2 plays an important role in numerous cellular processes including: proliferation, insulin secretion, senescence, glutamine metabolism and reactive oxygen species (ROS) homeostasis. I found that ME2 binds CoA tightly and, through mutation studies, identified the CoA-binding site of ME2. By screening various CoA species, I discovered a specific CoA molecule can dramatically increase ME2 enzymatic activity. Furthermore, I determined that the identified CoA molecule promotes ME2 tetramer formation and may have a role in relieving oxidative stress. This work demonstrates that cofactor-based chemical proteomic strategies work well to identify novel protein interactions. Expansion of this technique may prove useful in better understanding cellular regulation.