Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography

Nucleoside diphosphate (NDP) kinases display low specificity with respect t o the base moiety of the nucleotides and to the 3'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in th is paper the enzymatic analysis of a series of derivatives of thymidine dip hosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, a zido, and amino groups. With Dictyostelium NDP kinase, k(cat) decreases 15- 200-fold from 1100 s(-1) with TDP, and (k(cat)/K-m)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s(-1), depending on the substrate. The poor est substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best mod ified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. I n a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substr ate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP, (k (cat)/K-m)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor cat alytic efficiency of the modified nucleotides at a structural level, we det ermined the crystal structure of Dictyostelium NDP kinase complexed to 3'-f luoro-2',3'-dideoxyUDP at 2.7 Angstrom resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by t he 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to th e 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta -phosphate hydrogen bonds to Asn119 and moves away from the attacking N del ta of the catalytic His122, Since all anti-AIDS nucleoside drugs are modifi ed at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.