E. coli dihydrodipicolinate reductase exhibits unusual nucleotide specificity, with NADH being kinetically twice as effective as NADPH as u reductant as evidenced by their relative V/K values. To investigate the nature of the interactions which determine this specificity, we performed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes. The thermodynamic binding parameters for NADPH and NADH were determined to be K(d) = 2.12 μM, ΔG° = -7.81 kcal mol-1, ΔH° = -10.98 kcal mol-1, and ΔS° = -10.5 cal mol m-1 deg-1 and K(d) = 0.46 μM, ΔG° = -8.74 kcal mol-1 ΔH° = -8.93 kcal mol-1 and ΔS° = 0.65 cal mol-1 deg-1 respectively. The structures of DHPR complexed with these nucleotides have been determined at 2.2 Å resolution. The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38. Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contributions of individual groups on the nucleotide to the binding affinity and enthalpic and entropic components of the free energy of binding, ΔG°. Analogs lacking the 2'-phosphate group bound with a 4-5-fold higher affinity to the protein compared to their 2'-phosphate containing homologs. For all analogs, the total binding free energy can be shown to include compensating enthalpic and entropic contributions to the association constants. The entropy contribution appears to play a more important role in the binding of the nonphosphorylated analogs than in the binding of the phosphorylated analogs.
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