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

• Published in: Biochemistry (Easton) Vol. 34; no. 21; pp. 7207 - 7216
• Main Authors: Sarafianos, Stefanos G, Pandey, Virendra N, Kaushik, Neerja, Modak, Mukund J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.05.1995
• Subjects: Amino Acid Sequence; Base Sequence; Cations, Divalent; Cloning, Molecular; Deoxyribonucleotides - metabolism; Glutamine - metabolism; HIV Reverse Transcriptase; HIV-1 - enzymology; human immunodeficiency virus 1; Hydrogen-Ion Concentration; Molecular Sequence Data; Mutation; Nucleotidyltransferases - metabolism; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00021a036

In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.