D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis displays substantial substrate inhibition in the direction of NADH oxidation by its physiological substrate, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site. While addition of substrate at the active site is ordered, with HPAP binding before NADH, NADH can compete with the substrate for binding to the allosteric site and thereby eliminate the substrate inhibition. Fluorescence resonance energy transfer analysis of mutants with specific tryptophan residues converted to phenylalanine residues demonstrates that the main interaction of NADH with the enzyme, in the absence of substrate, is at the allosteric anion-binding site. This is further confirmed by mutations of basic residues at the anion-binding site which also demonstrates that these residues are necessary for inhibition by L-serine when it binds to the regulatory domain. This may indicate that a ligand must be bound to the anion-binding site for L-serine inhibition, providing a potential mechanism for low levels of activity in the presence of high levels of inhibitor.