Document 0882
 DOCN  M9590882
 TI    Glutamine 151 participates in the substrate dNTP binding function of
       HIV-1 reverse transcriptase.
 DT    9509
 AU    Sarafianos SG; Pandey VN; Kaushik N; Modak MJ; Department of
       Biochemistry and Molecular Biology, UMD-New Jersey; Medical School,
       Newark 07103, USA.
 SO    Biochemistry. 1995 May 30;34(21):7207-16. Unique Identifier : AIDSLINE
       MED/95284057
 AB    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.
 DE    Amino Acid Sequence  Base Sequence  Cations, Divalent  Cloning,
       Molecular  Deoxyribonucleotides/*METABOLISM  Glutamine/*METABOLISM
       Hydrogen-Ion Concentration  HIV-1/*ENZYMOLOGY  Molecular Sequence Data
       Mutation  Nucleotidyltransferases/METABOLISM  Reverse
       Transcriptase/GENETICS/*METABOLISM  Substrate Specificity  Support, U.S.
       Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).