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Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase

S G Sarafianos1, V N Pandey, N Kaushik

  • 1Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School, Newark 07103, USA.

Biochemistry
|May 30, 1995
PubMed
Summary

Glutamine 151 (Gln151) in HIV-1 RT is crucial for polymerase function. Mutating Gln151 to alanine (Q151A) severely impairs activity, affecting dNTP binding and catalysis, while Q151N is similar to wild-type.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • Human Immunodeficiency Virus type 1 Reverse Transcriptase (HIV-1 RT) is a key enzyme in viral replication.
  • Understanding the precise roles of individual amino acid residues in HIV-1 RT polymerase function is essential for developing antiviral therapies.

Purpose of the Study:

  • To elucidate the role of Glutamine 151 (Gln151) in the polymerase activity of HIV-1 RT.
  • To investigate the impact of specific mutations at the Gln151 position on enzyme kinetics, substrate binding, and catalytic efficiency.

Main Methods:

  • Site-directed mutagenesis was employed to create Gln151 to Asparagine (Q151N) and Gln151 to Alanine (Q151A) mutants of HIV-1 RT.
  • Enzyme kinetics were analyzed using various RNA and DNA templates.
  • Deoxynucleotide triphosphate (dNTP) binding affinities and substrate incorporation were assessed.

Related Experiment Videos

  • Photoaffinity cross-linking was used to evaluate DNA binding.
  • Divalent cation preferences were determined.

    • Main Results:

    • The Q151N mutant showed wild-type-like polymerase activity, while the Q151A mutant exhibited significantly reduced catalytic rates (kcat) with RNA templates (15-100 fold) and a moderate reduction with DNA templates (5 fold).
    • The Q151A mutant displayed unchanged dNTP affinity with RNA templates but an increased Km with DNA templates.
    • The Q151A mutant failed to perform the nucleotidyl transfer reaction and showed an altered preference for divalent cations (Mn2+ over Mg2+).
    • Mutations in the p66 subunit (p66Q151A/p51WT) recapitulated the impaired activity, suggesting Gln151's critical role in the catalytic subunit.

    Conclusions:

    • Gln151 is critically involved in both dNTP binding and the catalytic step of the nucleotidyl transfer reaction in HIV-1 RT.
    • The specific substitution at Gln151 significantly impacts the enzyme's substrate preference and catalytic mechanism.
    • Structural modeling suggests Gln151 interacts with the dNTP base moiety and potentially with Arg72 within the active site.