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.
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.
Kew
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Y
Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York 10461.
Qingbin
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S
Prasad
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VR
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
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