Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation

Abstract

Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a substrate (kcat/Km) as dTTP for the enzyme. In contrast, the four 2',3'-dideoxynucleoside 5'-triphosphates and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (d4TTP) were 6-30-fold less efficient substrates of the enzyme. The kcat values for all nucleotide analogues were similar, consistent with a kinetic model in which the steady-state rate-limiting step was dissociation of the template-primer from the enzyme [Reardon, J. E., & Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307]. The pre-steady-state kinetics of single-nucleotide incorporation were consistent with the kinetic model: [formula: see text] where E, TP, and dNTP represent reverse transcriptase, a defined-sequence DNA primed-RNA template, and 2'-deoxynucleoside 5'-triphosphate (or analogue), respectively. The dissociation constant (Kd1) for template-primer binding was 10 nM, and the estimated rate constants for association and dissociation of the enzyme.template-primer complex were 4 x 10(6) M-1 s-1 and 0.04 s-1, respectively. The dissociation constants (Kd2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6 microM, respectively. Thus, the differences in steady-state Km values were not due to differences in binding of the nucleotide analogues to the enzyme. In contrast, the rate-limiting step during single-nucleotide incorporation (kp) was sensitive to the structure of the nucleotide substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

The Motif D Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Critical for Nucleoside 5′-Triphosphate Selectivity

“…This discrimination was specific for a 3Ј-azido group. Indeed, the same experiment was performed using ddTTP instead of AZTTP, and similar results were obtained, although ddTTP is a less potent inhibitor of RT (30). There was no change in the order of increasing discrimination among various RT variants studied.…”

Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability

“…RT-primerkernplate association and rate constants have been determined in a number of studies using kinetic methods (Huber et al, 1989;Kati et al, 1992;Reardon, 1992;Beard & Wilson, 1993;Hsieh et al, 1993). For example, Kati et al (1992) and Hsieh et al (1993) measured the Kd for primer/template binding at 37 "C using the burst amplitude of single nucleotide incorporation and obtained values of 26 nM and 4.7 nM, respectively.…”

The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

“…The comparison of the activation properties of AZT and d4T illustrates well that the chemical nature of the modifications in the ribose moiety is critical for antiretroviral potency. For example, ddTTP is a potent inhibitor of RT in vitro (12), but ddT has limited therapeutic value.…”