Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase.
Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.
Painter
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GR
Division of Virology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Wright
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LL
Hopkins
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Furman
P A
PA
D001667:Binding, Competitive; D002384:Catalysis; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D005454:Fluorescence Polarization; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D009994:Osmolar Concentration; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
D003838:Deoxyadenine Nucleotides; D013942:Thymine Nucleotides; D014364:Tryptophan; D012194:RNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate