Binding of nucleotides to nucleoside diphosphate kinase: A calorimetric study

The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to d esign more effective antiviral nucleoside drugs (e.g., AZT). We carried our a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to char acterize the binding in terms of DeltaG degrees, DeltaH degrees and DeltaS degrees. Thermodynamic parameters of the interaction of ADP with the hexame ric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Mycococcus xanthus, at 20 degreesC, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, wh ereas the DeltaG degrees values obtained were similar due to enthalpy-entro py compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP-PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-am ino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimi dine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the st oichiometry of the interaction to be abnormal, with 9-12 binding sites/hexa mer. The presence of supplementary binding sites might have physiological i mplications.