Thermodynamic Investigation of Inhibitor Binding to 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase

Isothermal titration calorimetry (ITC) was used to investigate the binding of six inhibitors to 1-deoxy-Dxylulose-5-phosphate reductoisomerase (DXR), a target for developing novel anti-infectives. The binding of hydroxamate inhibitors to Escherichia coli DXR is Mg2+-dependent, highly endothermic (Delta H, 22.7-24.3 kJ/mol), and entropy-driven, while that of nonhydroxamate compounds is metal ion-independent and exothermic (Delta H, -19.4 to -13.8 kJ/mol), showing that hydration/dehydration of the enzyme metal ion binding pocket account for the drastic Delta H change. However, for DXRs from Plasmodium falciparum and Mycobacterium tuberculosis, the binding of all inhibitors is exothermic (Delta H, -24.9 to -9.2 kJ/mol), suggesting that the metal ion binding sites of these two enzymes are considerably less hydrated. The dissociation constants measured by ITC are well correlated with those obtained by enzyme inhibition assays (R-2 = 0.75). Given the rapid rise of antibiotic resistance, this work is of interest since it provides novel structural implications for rational development of potent DXR inhibitors.