REVERSE-TRANSCRIPTASE FROM HUMAN-IMMUNODEFICIENCY-VIRUS - A SINGLE TEMPLATE-PRIMER BINDING-SITE SERVES 2 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 dead-enylated 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 K(m), K(D), and k(cat) 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 k.t indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of K(m) and k(cat) 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 K(i) 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.