Analysis of the role of glutamine 190 in the catalytic mechanism of murine leukemia virus reverse transcriptase.
To determine the catalytic role of Gln(190), a member of the highly conserved LPQG motif in Moloney murine leukemia virus reverse transcriptase, we carried out site-directed mutagenesis of this residue to generate Q190N and Q190A. Both mutant proteins exhibited a significant loss in their polymerase and pyrophosphorolysis activities with a more pronounced effect noted with the Gln --> Asn substitution. The catalytic efficiencies of the mutants exhibited a 40-70-fold reduction with poly(rC) and poly(dC) templates in the presence of Mg(2+) and a 10-20-fold reduction with poly(rA) template in the presence of Mn(2+). Interestingly, the K(m) for NTP exhibited only a moderate 3-10-fold increase irrespective of the template-primer and the metal ion. Photoaffinity labeling of both the mutant and the WT enzymes exhibited an identical affinity for RNA.DNA and DNA.DNA template-primers. However, unlike the WT enzyme, the mutant enzymes exhibited a significantly reduced ability to catalyze the nucleotidyltransferase reaction on the covalently immobilized template-primer. An examination of the rate constants for the first and the second nucleotide for the mutant enzymes indicated dissimilar rates, indicating that Gln(190) may be involved in a rate-limiting, conformational change step both before and after the phosphodiester bond formation. Furthermore, the processivity of DNA synthesis by the mutant enzymes was decreased severely, which may result from the lower catalytic efficiency as well as translocation defect.
Jin
J
J
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103, USA.
Kaushik
N
N
Singh
K
K
Modak
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005973:Glutamine / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*
D005973:Glutamine; D012194:RNA-Directed DNA Polymerase