Tuberculosis (TB) is the leading cause of death from infectious disease, and new drugs are needed to treat emerging resistant TB infections. Using genetic, metabolomic, crystallographic, and recombinant enzyme assays we discovered a compound that kills Mycobacterium tuberculosis (Mtb) upon binding the active site of phosphopantetheinyl transferase (PptT). PptT is an essential enzyme for Mtb, and has been a desired drug target for many years. PptT uses coenzyme A (CoA) as a substrate to activate acyl carrier proteins (ACPs) by transferring phosphopantheine (Ppt) from CoA onto the ACP. ACPs then use the Ppt group for synthesis of lipids critical for Mtb’s cell wall and virulence. The compound did not fully abolish PptT activity, and we found that killing of Mtb by the inhibitor depended on the activity of a newly identified enzyme, a Ppt hydrolase (PptH) that can remove Ppt from ACPs. While PptH is highly conserved among mycobacteria, it’s opposing function to an essential process has no obvious utility to the cell. This activity was revealed by a new mechanism of antimicrobial resistance: loss of function in an enzyme (PptH) that opposes the action of the enzyme (PptT) targeted by the inhibitor. The opposing PptT/PptH reactions invite exploration of a regulatory pathway in CoA physiology, as the two enzymes oppose each other and may be involved in stress-response signaling.