title: NDP kinase reactivity towards 3TC nucleotides.
abstract: Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.
authors.affiliation: Unité de Régulation Enzymatique des Activités Cellulaires, CNRS FRE 2364 Institut Pasteur 25, rue du Dr Roux, 75724 Cedex 15, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Kreimeyer
authors.forename: B
authors.initials: B
authors.lastname: Schneider
authors.forename: R
authors.initials: R
authors.lastname: Sarfati
authors.forename: A
authors.initials: A
authors.lastname: Faraj
authors.forename: J P
authors.initials: JP
authors.lastname: Sommadossi
authors.forename: M
authors.initials: M
authors.lastname: Veron
authors.forename: D
authors.initials: D
authors.lastname: Deville-Bonne
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D003198:Computer Simulation; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology; D054306:Dideoxynucleotides; D006801:Humans; D007700:Kinetics; D019259:Lamivudine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; C417268:lamivudine triphosphate; D019259:Lamivudine; D003570:Cytidine Triphosphate; D009701:Nucleoside-Diphosphate Kinase