Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3'-OH of the incoming dTTP substrate.
Two previously identified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants, Q151N and V148I, are known to have reduced dNTP binding affinity but possess wild-type chemical catalysis rates. Structural modeling based on the crystal structure of the HIV-1 RT ternary complex with dTTP proposes that Q151N loses the interaction with the 3'-OH of the incoming dTTP and that V148I disrupts positioning of Q151 for this interaction. On the basis of this, we predicted that while wild-type (WT) HIV-1 RT would have decreased binding affinity to dTTP analogues lacking 3'-OH, compared to dTTP, the Q151N and V148I RT mutants should have decreased but similar affinity to both dTTP and dTTP analogues. Pre-steady-state kinetics on WT RT showed 14- and 53-fold higher K(d) values for the 3'-OH lacking ddTTP and acyTTP, compared to dTTP. In contrast, the Q151N and V148I mutants, which were predicted to have lost H-bonding interaction with the 3'-OH of dTTP, showed higher but similar K(d) values for dTTP, ddTTP, and acyTTP. Interestingly, the Q151N and V148I RTs bound to AZTTP approximately 12 and 18 times more tightly than to dTTP, respectively. Our structure modeling suggests that these RT mutants can interact with the azido moiety of AZTTP, which is 1.4 A longer than the 3'-OH of dTTP. The kinetic data presented in this report demonstrate the functional role of the Q151 residue in HIV-1 RT interaction with dTTP and its analogues containing chemical modifications at the 3'-C of the sugar moiety.
Jamburuthugoda
Varuni K
VK
Department of Biochemistry and Biophysics, University of Rochester, 601 Elmwood Avenue, Box 672, Rochester, New York 14642, USA.
Guo
Dongdong
D
Wedekind
Joseph E
JE
Kim
Baek
B
D000212:Acyclovir / Q000031:analogs & derivatives / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D054306:Dideoxynucleotides; D005973:Glutamine / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D014633:Valine / Q000235:genetics; D015215:Zidovudine / Q000031:analogs & derivatives / Q000737:chemistry
D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; D007532:Isoleucine; D005973:Glutamine; D017665:Hydroxyl Radical; D015215:Zidovudine; C025114:acyclovir triphosphate; C052873:zidovudine triphosphate; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D014633:Valine; C017216:2',3'-dideoxythymidine triphosphate; C024157:thymidine 5'-triphosphate; D000212:Acyclovir