3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

• Published in: The Journal of biological chemistry 1998-10, Vol.273 (44), p.28773-28778
• Main Authors: Schneider, Benoit, Xu, Ying Wu, Janin, Joël, Véron, Michel, Deville-Bonne, Dominique
• Format: Article
• Language: English
• Subjects: 5'-PHOPHOADENOSINE 3-PHOSPHATE; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADENOSINA; ADENOSINE; ADENOSINE 3'-PHOSPHATE 5'-PHOSPHOSULFATE; Animals; Binding Sites; CRYSTAL STRUCTURE; DICTYOSTELIUM; Dictyostelium - enzymology; DICTYOSTELIUM DISCOIDEUM; DISSOCIATION; ENZYME INHIBITORS; ENZYMIC ACTIVITY; Fluorescence; FOSFATOS (ESTERES); INHIBICION; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; INHIBITION; MOLECULAR CONFORMATION; Molecular Sequence Data; Molecular Structure; Nucleoside-Diphosphate Kinase - antagonists & inhibitors; Nucleoside-Diphosphate Kinase - metabolism; PDB/1BUX; PHOSPHATE (ESTER); PHOSPHATES (ESTERS); Phosphoadenosine Phosphosulfate - metabolism; Phosphoadenosine Phosphosulfate - pharmacology; Phosphorylation; TRANSFERASAS; TRANSFERASE; TRANSFERASES; X RAY CRYSTALLOGRAPHY
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.44.28773

Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3′-OH and the β-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3′-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3′-phosphate, 5′-phosphoadenosine 3′-phosphate (PAP) and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. TheKD of PAPS (10 μm) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-Å resolution. It shows a new mode of nucleotide binding at the active site with the 3′-phosphate of PAPS located near the catalytic histidine, at the same position as the γ-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.