Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase.

Abstract:

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.

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