Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides.
The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.
Schneider
B
B
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 1149, France.
Xu
Y W
YW
Sellam
O
O
Sarfati
R
R
Janin
J
J
Veron
M
M
Deville-Bonne
D
D
D000818:Animals; D019380:Anti-HIV Agents / Q000378:metabolism*; D001665:Binding Sites; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004023:Dictyostelium; D015224:Dideoxynucleosides / Q000378:metabolism*; D054306:Dideoxynucleotides; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D013942:Thymine Nucleotides / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
D019380:Anti-HIV Agents; D003854:Deoxyribonucleotides; D015224:Dideoxynucleosides; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate; D009701:Nucleoside-Diphosphate Kinase