The importance of monopole-monopole and monopole-dipole interactions on the binding of NADPH and NADPH analogues to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens effects of pH and ionic strength

NADPH binding to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02-0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values greater than 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate. The experimental data can be explained by a theoretical model in which monopole-monopole or monopole-dipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.