SITE-DIRECTED MUTAGENESIS OF ARGININE-72 OF HIV-1 REVERSE-TRANSCRIPTASE - CATALYTIC ROLE AND INHIBITOR SENSITIVITY

In order to determine the catalytic role of Arg(72) 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 k(cat) of the polymerase reaction was reduced by nearly 100-fold with poly(rA) tem plate, 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 k(cat) 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 Arg(72) 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 nueleotidyltransferase 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. Arg(72) is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.