NDP kinase reactivity towards 3TC nucleotides.
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.
Kreimeyer
A
A
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS FRE 2364 Institut Pasteur 25, rue du Dr Roux, 75724 Cedex 15, Paris, France.
Schneider
B
B
Sarfati
R
R
Faraj
A
A
Sommadossi
J P
JP
Veron
M
M
Deville-Bonne
D
D
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
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; C417268:lamivudine triphosphate; D019259:Lamivudine; D003570:Cytidine Triphosphate; D009701:Nucleoside-Diphosphate Kinase