Mechanism of HIV reverse transcriptase: enzyme-primer interaction as revealed through studies of a dNTP analog, 3'-azido-dTTP

Abstract

Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.cntdot.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et el. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].cntdot.oligo [d(T)] as template-primer. We find that 3''-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r-(A)].cntdot.oligo[d(T)]replication system. This suggests that 3''-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3''-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3''-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3''-azido-dTTP as substrate revealed that the Michaelis constant is 3 .mu.M. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex. This difference is consistent with the proposed kinetic scheme and is due primarily to the effect of enzyme-primer complex dissociation on the value of Km. The results enable calculation of rate values for the enzyme-poly[r(A)].cntdot.oligo[d(T)]association (kon = 2.6 .times. 108 M-1 s-1) and dissociation (koff = 39 s-1) and a KD value for dTTP binding (.apprx. 180 nM).