HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE - STEADY-STATE AND PRE-STEADY-STATE KINETICS OF NUCLEOTIDE INCORPORATION

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 (k(cat)/K(m)) 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 k(cat) 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: [GRAPHICS] 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 (K(d)1) 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 (K(d)2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6-mu-M, 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 (k(p)) was sensitive to the structure of the nucleotide substrate. The values of k(p) for dTTP, AZTTP, and ddfTP were 14, 5.4, and 0.41 s-1, respectively. The K(m) and k(cat)/K(m) values calculated from the values of K(d) and k(p) and the rate constants for dissociation of the enzyme template-primer complexes (k(off)) were in good agreement with the values obtained by steady-state kinetic analysis. Finally, there was no phosphorothioate elemental effect upon sulfur substitution at the alpha-phosphorus of dTTP. The results demonstrate that the substrate efficiency of nucleotide analogues was determined by the rate constant for phosphodiester bond formation or possibly by a conformational change preceding catalysis.