A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase
| Autor: | Jeremiah Hanes, Kenneth A. Johnson |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Kinetics
DNA-Directed DNA Polymerase Pyrophosphate 03 medical and health sciences chemistry.chemical_compound Genetics Humans Thymine Nucleotides heterocyclic compounds Nucleotide Enzyme kinetics Polymerase 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Nucleic Acid Enzymes 030306 microbiology Substrate (chemistry) DNA Polymerase gamma Mitochondria Diphosphates chemistry Biochemistry biology.protein Biophysics Reverse Transcriptase Inhibitors Thymidine Zidovudine |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkm695 |
| Popis: | Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena. |
| Databáze: | OpenAIRE |
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