Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263.
Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.
Martin
J L
JL
Division of Virology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Wilson
J E
JE
Furfine
E S
ES
Hopkins
S E
SE
Furman
P A
PA
D001483:Base Sequence; D003001:Cloning, Molecular; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000235:genetics*; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D011732:Pyridoxal Phosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D008239:Lysine