Analysis of the role of glutamine 190 in the catalytic mechanism of murineleukemia virus reverse transcriptase

To determine the catalytic role of Gln(190), a member of the highly conserv ed LPQG motif in Moloney murine leukemia virus reverse transcriptase, we ca rried out site-directed mutagenesis of this residue to generate Q190N and Q 190A, Both mutant proteins exhibited a significant loss in their polymerase and pyrophosphorolysis activities with a more pronounced effect noted with the Gin --> Asn substitution. The catalytic efficiencies of the mutants ex hibited a 40-70-fold reduction with poly(rC) and poly(dC) templates in the presence of Mg2+ and a 10-20-fold reduction with poly(rA) template in the p resence of Mn2+. Interestingly, the K-m for NTP exhibited only a moderate 3 -10-fold increase irrespective of the template-primer and the metal ion. Ph otoaffinity labeling of both the mutant and the WT enzymes exhibited an ide ntical affinity for RNA DNA and DNA DNA template-primers. However, unlike t he WT enzyme, the mutant enzymes exhibited a significantly reduced ability to catalyze the nucleotidyltransferase re action on the covalently immobili zed template-primer. An examination of the rate constants for the first and the second nucleotide for the mutant enzymes indicated dissimilar rates, i ndicating that Gln190 may be involved in a rate-limiting, conformational ch ange step both before and after the phosphodiester bond formation. Furtherm ore, the processivity of DNA synthesis by the mutant enzymes was decreased severely, which may result from the lower catalytic efficiency as well as t ranslocation defect.