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

Y. Kew, S. Qingbin, V. R. Prasad

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32 Scopus citations

Abstract

The E89C 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 (p66(wt)/p51m and p66m/p51(wt)), and the mutant enzyme (p66m/p51m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66m/p51m) displayed a higher V(max), a higher K(m) for 2'-deoxythymidine triphosphate, and a higher K(i) for 2',3'- dideoxythymidine triphosphate than the wild type enzyme. The increased K(m) and K(i) 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 (p66(wt)/p51(wt) and p66(wt)/p51m) displayed a preference for magnesium. Our results indicate that p66 plays a dominant role in deoxynucleotide triphosphate substrate recognition (K(m)), nucleoside analog sensitivity (K(i)), and magnesium preference. However, the increased V(max) 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 - Jan 1 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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