Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (DeltaH = -44.5 +/- 0.1 kJ mol(-1)) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (DeltaH = -2.8 +/- 0.1 kJ mol(-1)) formed through the reduction of the intermediate pyruvyl-Schiff-base complex. The binding interaction of (S)-lysine was characterised as a cooperative event in which an entropic pre-organisation step (TDeltaS = 17.6 +/- 1.1 kJ mol(-1)) precedes a secondary enthalpic association (DeltaH = -21.6 +/- 0.2 kJ mol(-1)). This allosteric association was determined to be of a mixed competitive nature in which heterotropic ligand cooperativity was observed to subtly influence the binding events. These results offer new insights into the inhibition of this enzyme, a validated antibiotic target.