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Active site studies of human immunodeficiency virus reverse transcriptase.

L L Mitchell1, B S Cooperman

  • 1Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323.

Biochemistry
|August 25, 1992
PubMed
Summary

Investigating human immunodeficiency virus reverse transcriptase (HIV1-RT) active sites revealed key residue roles. Arginine at position 277 is crucial, while cysteine 162

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • Human immunodeficiency virus reverse transcriptase (HIV1-RT) is a critical enzyme for viral replication.
  • Understanding the HIV1-RT active site is essential for developing antiviral therapies.
  • Previous studies have utilized chemical probes to investigate enzyme active sites.

Purpose of the Study:

  • To probe the active site of HIV1-RT using group-specific chemical reagents.
  • To identify key amino acid residues involved in enzyme activity and primer-template binding.
  • To differentiate the inhibition mechanisms of specific reagents on different HIV1-RT variants.

Main Methods:

  • Inactivation assays using phenylglyoxal (PG), N-ethylmaleimide (NEM), and pyridoxal 5'-phosphate (PLP).
  • Protection experiments with primer-template to assess reagent binding site.
  • Comparative analysis of inhibition on different HIV1-RT variants (NY5-HIV1-RT and LAV-HIV1-RT).

Main Results:

  • Arginine-specific PG inactivated HIV1-RT, with protection by primer-template, localizing a potential active site arginine to position 277.
  • Sulfhydryl-modifying NEM completely inhibited NY5-HIV1-RT (C162) but not LAV-HIV1-RT (S162), suggesting C162's role.
  • Lysine-specific PLP acted as a noncompetitive inhibitor against primer-template and dTTP, distinct from other RTs.

Conclusions:

  • Arginine 277 is likely located within or near the active site of HIV1-RT's polymerase domain.
  • Cysteine 162 may be at or near the active site or allosterically interact with primer-template but is not essential for activity.
  • The noncompetitive inhibition by PLP differentiates HIV1-RT from other reverse transcriptases regarding lysine residue involvement.

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