Reverse transcriptase from human immunodeficiency virus: a single template-primer binding site serves two physically separable catalytic functions.
The binding of substrates to recombinant reverse transcriptase from human immunodeficiency virus (HIV) and the natural enzyme from avian myeloblastosis virus (AMV) has been examined by analyzing both the ribonuclease H and the RNA-dependent DNA polymerase activities. With 3'-end-labeled globin mRNA hybridized to (dT)15 as the substrate in the ribonuclease H reaction, the enzymes partially deadenylated the mRNA in a distributive manner. Under these conditions, there was a rapid initial burst followed by a prolonged, but much slower, steady-state rate. The biphasic reaction made possible determinations of kinetic constants as follows: values for Km, KD, and kcat were, respectively, 27 nM, 11 nM, and 5 x 10(-3) s-1 for the HIV enzyme and 30 nM, 9 nM, and 5 x 10(-3) s-1, respectively, for the avian enzyme. These constants were used to derive other parameters: The rate of association of the template-primer with reverse transcriptase was approximately 2 x 10(5) M-1 s-1, and the rate of dissociation was approximately 2 x 10(-3) s-1, regardless of the source of the enzyme. The rate of release of the product was essentially equivalent to the value of kcat indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of Km and kcat of approximately 6 nM and approximately 2.5 s-1, respectively, for the human enzyme, and approximately 10 nM and approximately 2 s-1, respectively, for the avian enzyme were observed. The interaction of substrates with HIV reverse transcriptase was characterized further with the aid of ribonucleoside-vanadyl complexes. These complexes inhibited the polymerase and ribonuclease H activities of the enzyme competitively with respect to globin mRNA.(dT)15. Values of Ki ranging from 1 to 3 mM were obtained. With respect to deoxyribonucleoside triphosphate substrates in the polymerase reaction, mixed inhibition was observed. Deoxyribonucleoside triphosphates had no effect on kinetic parameters governing the ribonuclease H activity of the HIV enzyme but apparently facilitated the formation of active enzyme. These data fit a model in which one template-primer binding site serves both the polymerase and the ribonuclease H catalytic sites.
Krug
M S
MS
Section on Genes and Gene Products, National Cancer Institute, NIH, Bethesda, MD 20892.
Berger
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SL
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D005914:Globins / Q000235:genetics; D006678:HIV / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; C027903:oligo (dT); D014638:Vanadates; D012313:RNA; D005914:Globins; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H