Substrate binding in human immunodeficiency virus reverse transcriptase. An analysis of pyridoxal 5'-phosphate sensitivity and identification of lysine 263 in the substrate-binding domain.
Human immunodeficiency virus reverse transcriptase (HIV-RT) exhibits a strong sensitivity to pyridoxal 5'-phosphate (PLP), a substrate-binding site directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of HIV-RT with PLP followed by sodium borohydride reduction of the enzyme-PLP adduct results in irreversible inactivation of polymerase activity while RNase H activity associated with HIV-RT is minimally affected. Kinetic studies indicate that the PLP inhibition is complex. Yet one of the sites of PLP action appears to be involved in the process of dNTP binding as judged by (a) competitive mode of inhibition and (b) blockage of PLP into enzyme protein by the addition of substrate dNTP. Furthermore, this site is the only PLP reactive site which is accessible to borohydride reduction. Comparative tryptic peptide mapping of enzyme treated with PLP under a variety of conditions permitted the identification of a PLP reactive site containing peptide. Furthermore, reactivity of this site was also blocked by inclusion of substrate dNTP and appropriate template-primer. The amino acid composition and sequence analysis of this peptide showed that a lysine residue present at position 263 in the primary amino acid sequence of HIV-RT is the site of PLP reactivity. We therefore conclude that lysine 263 serves as an important part of the dNTP-binding domain in HIV-RT.
Basu
A
A
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103-2757.
Tirumalai
R S
RS
Modak
M J
MJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016914:Ribonuclease H
D000596:Amino Acids; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; D008239:Lysine