Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity.
In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.
Sarafianos
S G
SG
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, Newark 07103, USA.
Pandey
V N
VN
Kaushik
N
N
Modak
M J
MJ
D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011061:Poly A / Q000378:metabolism; D011066:Poly C / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity
D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D011061:Poly A; D011066:Poly C; D001120:Arginine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase