Binding of Nucleotides to Nucleoside Diphosphate Kinase: A Calorimetric Study ‡ Laura Cervoni, §,| Ioan Lascu, ⊥ Yingwu Xu, # Philippe Gonin, ⊥ Michael Morr, 3 Mohamed Merouani, ⊥ Joel Janin, # and Anna Giartosio* ,§,| Istituto Pasteur Fondazione Cenci Bolognetti and Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, UniVersita ` di Roma “La Sapienza”, 5 P. le Aldo Moro, 00185 Roma, Italy, Institut de Biochimie et Ge ´ ne ´ tique Cellulaires (UMR 5095), Centre National de la Recherche Scientifique et UniVersite ´ de Bordeaux-2, 33077 Bordeaux, France, Laboratoire d'Enzymologie et Biochimie Structurales UPR9063 CNRS, 91198 Gif-sur-YVette, France, and Gesellschaft fu ¨ r Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany ReceiVed October 18, 2000; ReVised Manuscript ReceiVed January 22, 2001 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 ∆G°, ∆H° and ∆S°. 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 °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 ∆G° 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. Nucleoside diphosphate (NDP) 1 kinases catalyze the reversible phosphorylation of nucleoside diphosphates by nucleoside triphosphates, via a ping-pong mechanism involv- ing a phosphohistidine intermediate (1-5). These ubiquitous enzymes play a key role in the metabo- lism of nucleotides, their principal function being the synthesis of (deoxy)nucleotide triphosphates from ATP and (deoxy)nucleotide diphosphates. Thus, for example, they supply GTP to G-proteins in signal transduction and sub- strates to nucleic acid synthesis (6, 7). Several crystal structures have been obtained for free NDP kinase (8-11), the phosphorylated intermediate (12), the enzyme complexed with ADP (9, 10), with GDP (13), with TDP (14), with 3′-azido-3′-dTDP and 3′-F-2′,3′-dideoxy UDP (15), and the enzyme complexed with ADP and AlF 3 ,a putative transition state analogue (16). These structures are reviewed in Janin et al. (17). The contacts between the nucleotides and the protein are remarkably conserved in enzymes from different sources, from bacteria to humans, as is overall protein subunit structure. Eukaryotic enzymes are hexameric, whereas some bacterial enzymes are tet- rameric (9). No major conformational differences were observed when the structures of the free, phosphorylated and nucleotide-bound enzymes were superimposed, except for a movement of the RA-R2 helices required for nucleotide binding. The base moiety lies in a hydrophobic crevice and makes no specific polar interactions with the protein. The substrate 2′-OH is not required for binding or catalysis, being replaced by a water molecule. This fits well with the broad specificity of NDP kinases, necessary for in vivo function. The 3′-OH is within hydrogen-bonding distance of the oxygen bridging the phosphates  and γ of the substrate NTP. Rapid-mixing studies (18) and steady-state experiments (5, 19) have shown that the absence of the ribose 3′-OH ‡ Atomic coordinates have been deposited in the Protein Data Bank (filename 1hiy). * To whom correspondence should be addressed. Phone: +39 06 49910576. Fax: +39 06 4440062. E-mail: anna.giartosio@uniroma1.it. § Istituto Pasteur Fondazione Cenci Bolognetti. | Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”. ⊥ Institut de Biochimie et Ge ´ne ´tique Cellulaires. # Laboratoire d'Enzymologie et Biochimie Structurales UPR9063 CNRS. 3 Gesellschaft fu ¨r Biotechnologische Forschung mbH. 1 Abbreviations: NDP, nucleoside diphosphate; D.d., Dictyostelium discoideum; M.x., Myxococcus xanthus; AZT-DP, 3′-azido-3′-deoxy- thymidine diphosphate; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2- ethane-sulfonic acid); 3′-NH2-ADP, 3′-amino-3′-deoxyADP; AMP-PNP, 5′-adenylylimido-diphosphate; DSC, differential scanning calorimetry; ITC, isothermal titration calorimetry. N 1 TP + E-His S N 1 DP + E-His-P (1a) E-His-P + N 2 DP S E-His + N 2 TP (1b) N 1 TP + N 2 TP S N 1 DP + N 2 TP (1) 10.1021/bi002432s CCC: $20.00 © xxxx American Chemical Society PAGE EST: 6.5 Published on Web 00/00/0000