Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.
The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K(m) values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.
Zakharenko
A L
AL
Novosibirsk Institute of Bioorganic Chemistry, pr. Lavrent'eva 8, Novosibirsk-90, 630090 Russia. sashaz@niboch.nsc.ru
Kolpashchikov
D M
DM
Khodyreva
S N
SN
Lavrik
O I
OI
Menéndez-Arias
L
L
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D003856:Deoxyuracil Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D054306:Dideoxynucleotides; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry*; D010777:Photochemistry / Q000379:methods; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D014500:Uracil Nucleotides / Q000737:chemistry / Q000378:metabolism
C119021:5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D019920:Photoaffinity Labels; D014500:Uracil Nucleotides; C027078:deoxyuridine triphosphate; D014443:Tyrosine; D008345:Manganese; C063034:2',3'-dideoxyuridine-5'-triphosphate; D014364:Tryptophan; D054303:HIV Reverse Transcriptase; D008274:Magnesium; D000409:Alanine