Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition

Abstract

A study of steady-state kinetics of polymerization by purified human immunodeficiency virus DNA polymerase (reverse transcriptase) has been conducted. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer, and dTTP as nucleotide substrate. The substrate initial velocity patterns point to an ordered mechanism with template-primer adding first. Product inhibition kinetics with either pyrophosphate or phosphonoformate are consistent with this mechanism. The human immunodeficiency virus reverse transcriptase acts processively in this replication system, but exhibits some probability of terminating after each dTMP addition to the nascent chain. The probability of terminating was approximately 20-fold higher after the first dTMP addition than after subsequent additions. With this information on the mode of polymerization, appropriate kinetic models and steady-state rate equations are discussed. In further studies, we found that a heterologous polynucleotide, poly(rC), is a potent inhibitor of the enzyme. The pattern of this inhibition is uncompetitive against template-primer, suggesting that interaction with free enzyme is not the mechanism of the inhibition.