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

Yvonne Kew, Song Qingbin, Vinayaka R. Prasad

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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.

Original languageEnglish (US)
Pages (from-to)15331-15336
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number21
StatePublished - May 27 1994

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HIV Reverse Transcriptase
Mutagenesis
Divalent Cations
Nucleosides
Kinetics
Enzymes
Magnesium
RNA-Directed DNA Polymerase
Kinetic parameters
Dimers
Thymidine
Cations
HIV-1
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

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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.",
author = "Yvonne Kew and Song Qingbin and Prasad, {Vinayaka R.}",
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T2 - Contribution of p66 and p51 Subunits to Nucleoside Analog Sensitivity, Divalent Cation Preference, and Steady State Kinetic Properties

AU - Kew, Yvonne

AU - Qingbin, Song

AU - Prasad, Vinayaka R.

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N2 - 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.

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