Mutational analysis of Lys(65) of HIV-1 reverse transcriptase

Amino acid Lys(65) is part of the highly flexible beta 3-beta 4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-I) reverse transcriptase (RT). Recent crystal data show that the eps ilon-amino group of Lys(65) interacts with the gamma-phosphate of the bound deoxynucleoside triphosphate ('dNTP') substrate [Huang, Chopra, Verdine an d Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys(65), We have used site-specific mutagenesis to gene rate RT with a variety of substitutions at this position, including K65E, K 65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys(65) in RT is s ubstituted with an amino acid other than arginine the enzyme exhibits drama tic decreases in the binding affinity (K-m) for all dNTP substrates, in RT catalytic efficiency (k(cat)/K-m) and in the mutant enzyme's ability to car ry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH op timum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 f or the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molec ular modelling studies show that mutations of Lys(65) do not affect the geo metry of the loop's alpha-carbon backbone, but rather lead to changes in po sitioning of the side chains of residues Lys(70) and Arg(72). In particular , Glu in K65E can form a salt bridge with Arg(72) leading to the diminution of the latter residue's interaction with the alpha-phosphate of the dNTP r esidue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme . Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sen sitive to all dideoxynucleoside triphosphate ('ddNTP') inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddC TP and ddATP only. This implies that mutations at position 65 in HIV-I RT i nfluence the nucleotide-binding specificity of the enzyme.