J. Biol. Chem., Vol. 267, Issue 35, 25019-25024, 12, 1992
Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates
NA Van Draanen, SC Tucker, FL Boyd, BW Trotter and JE Reardon
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
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.
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