Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the Ki for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena. The change in Ki has an average pK of ∼5.8 and involves two functional groups that are protonated and two functional groups that are unprotonated for optimal substrate inhibition to occur. Mutagenesis of positively charged amino acid residues at a putative anion binding site previously revealed by the x-ray structure, produces significant changes in the pH-dependent profile of substrate inhibition. Several single residue mutations eliminate the dual pH optima by reducing substrate inhibition between pH 5 and 7 and a triple mutation was identified that eliminates the substrate inhibition altogether. The mutagenesis data support the conclusion that the anion binding site represents a new allosteric site for the control of enzyme activity and functions in a novel mechanism for substrate inhibition.