HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE - CONTRIBUTION OF MET-184 TO BINDING OF NUCLEOSIDE 5'-TRIPHOSPHATE

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanin e (M184A) or valine (M184V), and steady-state and pre-steady-state kin etic constants were determined. The K-m values of M184A RT for dNTPs w ere larger than those of wt RT of RNA-directed synthesis; the k(cat) v alues of M184A RT for processive or distributive synthesis were simila r. In contrast to M184A RT, the K-m and k(cat) values of M184V RT for dNTP substrates were similar to those of wt RT. The K-i values of M184 V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relati ve to wt RT for both RNA- and DNA-directed synthesis. The K-d and k(p) values of wt RT and M184V RT for dCTP and 1-[2-(hydroxy-methyl)-1,3-o xathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimate d from pre-steady-state kinetics for single nucleotide incorporation. The K-d value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the k(p) values of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conse rved YMDD region of wt RT participates in the binding of the nucleosid e (analog) 5'-triphosphate.