Pre-steady State of Reaction of Nucleoside Diphosphate Kinase with Anti-HIV Nucleotides
| Autor: | Michel Véron, Dominique Deville-Bonne, Ying Wu Xu, Joël Janin, Benoit Schneider, Robert Sarfati, Olivier Sellam |
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| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular Anti-HIV Agents Stereochemistry Deoxyribonucleotides Biochemistry Structure-Activity Relationship chemistry.chemical_compound Reaction rate constant Animals Thymine Nucleotides Dictyostelium Nucleotide Phosphorylation Binding site Molecular Biology chemistry.chemical_classification Binding Sites biology Chemistry Kinase Active site Cell Biology Dideoxynucleosides Nucleoside-diphosphate kinase Dissociation constant Kinetics Dideoxynucleotide Nucleoside-Diphosphate Kinase Mutagenesis Site-Directed biology.protein Zidovudine Dideoxynucleotides |
| Zdroj: | Journal of Biological Chemistry. 273:11491-11497 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.273.19.11491 |
| Popis: | The pre-steady-state reaction ofDictyostelium 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 K S 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 inK D. 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 ink cat values. |
| Databáze: | OpenAIRE |
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