Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.

In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state ternary complex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 were completely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions, respectively. A molecular model of the ternary complex based on these results is also presented.

Glutamine-151 of HIV-1 RT has been shown to be a catalytically important residue through the characterization of its mutant phenotype Glu151Ala (Sarafianos et al., 1995a). To further understand the role of this residue, we have extended this analysis to include polymerization on natural RNA template in addition to DNA template. We find that Q151A mutant exhibited a severe reduction in the polymerase activity without any significant effect on the affinity for dNTP substrate. Unlike DNA-directed reactions, the rate-limiting step for RNA-directed reactions does not appear to be either at the dNTP binding step or the chemical step. Analysis of the products formed on natural heteromeric HIV-genomic RNA template annealed with an 18-mer DNA primer with a sequence complementary to the primer binding site (PBS) has shown that addition of nucleotides is nonlinear with time since the enzyme appears to stall on the RNA template following the incorporation of the first nucleotide. The Q151A mutant was found to be nearly devoid of pyrophosphorolytic activity on a RNA-PBS template-primer. Similar properties have been previously reported for a mutant of R72 (R72A) of HIV-1 RT (Sarafianos et al., 1995b). However, R72 was implicated in stabilizing the transition state ternary complex before and after the phosphodiester bond formation (Kaushik et al., 1996; Sarafianos et al., 1995b). Our results with Q151A suggest that the side chain of Q151 may help stabilize the side chain of R72, and the loss of pyrophosphorolysis activity observed with the Q151 mutant may be the indirect manifestation of this stabilizing effect on R72. These observations point to the functional interdependence of residues Q151 and R72 in the polymerase function of the enzyme. An analysis of the 3D model structure of HIV-1 RT bound to DNA-DNA and RNA-DNA template-primer reveals that the guanidine hydrogen of R72 seems to stabilize Q151 by hydrogen bonding with its amide oxygen. A systematic conformational search of the side chain of Q151 also suggests a stable orientation where its specific interaction with the base of the RNA template may aid in stabilizing it.