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 (p66^wt/p51^m and p66^m/p51^wt), and the mutant enzyme (p66^m/p51^m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66^m/p51^m) 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/p51^m) 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 (p66^m/p51^m) appeared to be determined by both of the subunits.