A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.
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.
Hanes
Jeremiah W
JW
Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.
Johnson
Kenneth A
KA
D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism; D015215:Zidovudine / Q000378:metabolism*
D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D015215:Zidovudine; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate