Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates.
beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism*
D003854:Deoxyribonucleotides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C034970:bacteriophage T7 induced DNA polymerase; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate