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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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