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 β-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analognes 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 K(D) of PAPS (10 μM) is three times lower than the K(D) of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a K(I) 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.