INTERACTION OF PYRIDINE-NUCLEOTIDE SUBSTRATES WITH ESCHERICHIA-COLI DIHYDRODIPICOLINATE REDUCTASE - THERMODYNAMIC AND STRUCTURAL-ANALYSIS OF BINARY COMPLEXES

E. coli dihydrodipicolinate reductase exhibits unusual nucleotide spec ificity, with NADH being kinetically twice as effective as NADPH as a reductant as evidenced by their relative V/K values. To investigate th e nature of the interactions which determine this specificity, we perf ormed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes. The thermodynamic bi nding parameters for NADPH and NADH were determined to he K-d = 2.12 m u M, Delta G degrees = -7.81 kcal mol(-1), Delta H degrees = -10.98 kc al mol(-1), and Delta S degrees = -10.5 cal mol(-1) deg(-1) and K-d = 0.46 mu M, Delta G degrees = -8.74 kcal mol(-1), Delta H degrees -8.93 kcal mol(-1), and Delta S degrees = 0.65 cal mol(-1) deg(-1), respect ively. The structures of DHPR complexed with these nucleotides have be en determined at 2.2 Angstrom resolution. The 2'-phosphate of NADPH in teracts electrostatically with Arg39, while in the NADH complex this i nteraction is replaced by hydrogen bonds between the 2' and 3' adenosy l ribose hydroxyls and Glu38. Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contribu tions of individual groups on the nucleotide to the binding affinity a nd enthalpic and entropic components of the free energy of binding, De lta G degrees. Analogs lacking the 2'-phosphate group bound with a 4-5 -fold higher affinity to the protein compared to their 2'-phosphate co ntaining 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.