HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE - 3'-AZIDODEOXYTHYMIDINE 5'-TRIPHOSPHATE INHIBITION INDICATES 2-STEP BINDING FOR TEMPLATE-PRIMER

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism, After template-primer (T . P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T . P binary complex (RT(T . P)). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T . P (for single-nucleotide incorporation), To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T . P system, poly(rA)-oligo(dT)(16). Inhibition of processive deoxythymidine monophosphate in corporation by these chain-terminating substrates was also examined Because AZTTP is a substrate, its K-m should be equivalent to K-i, and since K-m for AZTTP should be influenced by the dissociation rate constant for RT(T . P), we examined the effect of altering RT(T .) P dissociation on AZTTP kinetic parameters, The dissociation rate constant was modulated by making use of different T . P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T . P binding, As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)(16) was biphasic, with a burst followed by a slower steady-state phase representing k(cat) (0.42 min(-1)) which was similar to the rate constant for RT(T . P) dissociation. Additionally, K-m for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (K-i,K-AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar, However, a simple correlation between the RT(T . P) dissociation rate constant and K-i,K-AZTTP was not observed, These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RT(T . P) exist which can limit catalysis, The results are discussed in the context of a two-step binding reaction for T . P where the binary RT(T . P) complex undergoes an isomerization before binding of the deoxynucleotide substrate.