Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide.
Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).
Majumdar
C
C
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Stein
C A
CA
Cohen
J S
JS
Broder
S
S
Wilson
S H
SH
D001667:Binding, Competitive; D004261:DNA Replication; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D013698:Templates, Genetic; D013873:Thionucleotides / Q000494:pharmacology*
D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D013873:Thionucleotides; C059536:Sd(C)28; D012194:RNA-Directed DNA Polymerase