3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.
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 beta-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. The KD of PAPS (10 microM) 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-A 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 gamma-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.
Schneider
B
B
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1773, Institut Pasteur, Paris, France.
Xu
Y W
YW
Janin
J
J
Véron
M
M
Deville-Bonne
D
D
D000818:Animals; D001665:Binding Sites; D004023:Dictyostelium / Q000201:enzymology; D005453:Fluorescence; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009701:Nucleoside-Diphosphate Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation
D010724:Phosphoadenosine Phosphosulfate; D009701:Nucleoside-Diphosphate Kinase