Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase.
Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49). An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer. Similar values were obtained with the homodimeric and the virus-encoded enzymes. When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM. Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used. Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit. Furthermore, UV crosslinking experiments with [gamma-32P]dTTP showed crosslinking only to the p66 subunit. Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase.
Furman
P A
PA
Division of Virology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.
Painter
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G
Wilson
J E
JE
Cheng
N
N
Hopkins
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D018894:Reverse Transcriptase Inhibitors; D013698:Templates, Genetic; D013936:Thymidine / Q000494:pharmacology
D003854:Deoxyribonucleotides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D000255:Adenosine Triphosphate; D012194:RNA-Directed DNA Polymerase; D013936:Thymidine