Binding of Nucleotides to Nucleoside Diphosphate Kinase:  A Calorimetric Study

Abstract

The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, 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, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy 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'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications.

The advantage of channeling nucleotides for very processive functions

“…As the cellular concentration of ATP is much higher than that of other NTPs, the reversible NDPK reaction is driven towards phosphoryl transfer from ATP to GDP, CDP, or UDP to form their corresponding NTPs. Although NDPKs are considered non-specific with respect to the base moiety of acceptor nucleotides, guanine nucleotides are their best substrates, whereas cytosine nucleotides are the poorest in terms of both K m and k cat 18, 19 .…”

Receptor activation regulates cortical, but not vesicular localization of NDP kinase

“…NDP kinases bind nucleotides with affinities in the range of 10-200 µM, and guanine nucleotides are somewhat preferred over other substrates (Schaertl et al, 1998;Cervoni et al, 2001;Schneider et al, 2002). Therefore, the simplest explanation for the preferential release of NDP kinase from the MT/Ves fraction by GTP is that it involves binding of the nucleotide directly to its catalytic site.…”

“…However, if the release is mediated by the catalytic site of NDP kinase, the thiophosphate analogs GTPγS and GDPβS, which are substrates for NDP kinases (Schaertl et al, 1998), should behave similarly to GTP. Imidodiphosphate analogs bind to NDP kinases with low affinity and are not substrates (Cervoni et al, 2001), so GMP-PNP should have modest effects, if any, on the retention of NDP kinase by the MT/Ves fraction. As seen in Fig.…”

Nucleotide Binding to Nucleoside Diphosphate Kinases: X-ray Structure of Human NDPK-A in Complex with ADP and Comparison to Protein Kinases

“…On the other hand, NMPs, which are inhibitors, have b < 2 308, g < 2 608, and there is a group of NDP kinase ligands that has b < 1408, g < 608, defining region B in Figure 7(A). This group includes 3 0 -amino ADP, a very poor substrate, 16 and conformers of ADP observed only in complexes where two conformations coexist (1NDP, 1NLK).…”