Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity.

The Journal of Biological Chemistry
Stefan G SarafianosM J Modak

Abstract

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 bindi...Continue Reading

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Citations

Mar 21, 2000·Medicinal Research Reviews·H JonckheereE De Clercq
Apr 27, 2013·Journal of Computer-aided Molecular Design·Kamil Maláč, Ivan Barvík
May 5, 2000·The Biochemical Journal·N Sluis-CremerM A Parniak
Jun 11, 2009·The Journal of Biological Chemistry·Egor P TchesnokovMatthias Götte
Sep 8, 2012·The Journal of Biological Chemistry·Kamalendra SinghStefan G Sarafianos
Aug 16, 2000·The EMBO Journal·M C MillerK Collins
Jan 15, 2016·Nucleic Acids Research·Kamal K SharmaYves Mély
Jul 21, 2009·Antiviral Research·Luis Menéndez-Arias
Jun 5, 2010·Viruses·Antonio J Acosta-Hoyos, Walter A Scott
Dec 29, 2020·Nucleic Acids Research·Sumit HandaPartho Ghosh
Jan 29, 2021·Chemical Reviews·Maria E CilentoStefan G Sarafianos
Jan 1, 2008·Bioconjugate Chemistry·Joseph F Krebs, Anilkumar R Kore
Aug 24, 2021·European Journal of Medicinal Chemistry·Weijie GuSteven De Jonghe

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