title: Subunit-selective mutagenesis of Glu-89 residue in human immunodeficiency virus reverse transcriptase. Contribution of p66 and p51 subunits to nucleoside analog sensitivity, divalent cation preference, and steady state kinetic properties.
abstract: The E89G alteration in the human immunodeficiency virus type 1 reverse transcriptase has been shown to confer resistance to nucleoside analogs and a loss of magnesium cation preference (Prasad, V.R., Lowy, I., De Los Santos, T., Chiang, L., and Goff, S.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11363-11367. The wild type reverse transcriptase heterodimer, chimeric reverse transcriptases that contain the E89G alteration in one of the subunits (p66wt/p51m and p66m/p51wt), and the mutant enzyme (p66m/p51m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66m/p51m) displayed a higher Vmax, a higher Km for 2'-deoxythymidine triphosphate, and a higher Ki for 2',3'-dideoxythymidine triphosphate than the wild type enzyme. The increased Km and Ki values were observed only when a heterodimer contained the alteration in the p66 subunit. Tests for divalent cation requirement showed that only the dimers containing the wild type p66 (p66wt/p51wt and p66wt/p51m) displayed a preference for magnesium. Our results indicate that p66 plays a dominant role in deoxynucleotide triphosphate substrate recognition (Km), nucleoside analog sensitivity (Ki), and magnesium preference. However, the increased Vmax displayed by the mutant enzyme (p66m/p51m) appeared to be determined by both of the subunits.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York 10461.
authors.forename: Y
authors.initials: Y
authors.lastname: Kew
authors.forename: S
authors.initials: S
authors.lastname: Qingbin
authors.forename: V R
authors.initials: VR
authors.lastname: Prasad
mesh_terms: D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D017931:DNA Primers; D054306:Dideoxynucleotides; D017245:Foscarnet / Q000494:pharmacology; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D017931:DNA Primers; D054306:Dideoxynucleotides; D005971:Glutamates; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D017245:Foscarnet; D018698:Glutamic Acid; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate