Factors affecting the dimerization of the p66 form of HIVâ1 reverse transcriptase
2001
Cabodevilla, Jesús F. | Odriozola, Leticia | Santiago, Esteban | MartínezâIrujo, Juan J.
The association and dissociation of the homodimeric p66/p66 form of HIVâ1 reverse transcriptase were investigated. The effects on the dimerization process of different salt concentrations, pH and the presence of a template/primer and nucleotide substrates were monitored by measuring polymerase activity and analytical sizeâexclusion HPLC. At submicromolar concentrations of enzyme and physiological salt concentrations, most of the enzyme exists in the inactive monomeric form. Increasing NaCl concentration from 0.05 to 1âm decreased the equilibrium dissociation constant from 2.0 to 0.34âµm. Analysis of the kinetics of the dimerization process indicated it followed a twoâstep mechanism, with rapid initial association of the two subunits to form an inactive homodimer followed by a slow isomerization step rendering the active enzyme form. The presence of poly(rA)/dT20 decreased the equilibrium dissociation constant of the homodimer about 30âfold, while the addition of 5âµm dTTP had no effect. The kinetics of the process showed that the template/primer favored dimerization by binding to the inactive homodimer and promoting its isomerization to the active form. These results were confirmed by analyzing the reverse reaction, i.e. the dissociation of the enzyme, by dilution in a lowâionicâstrength buffer. The results suggest that binding of immature HIVâ1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.
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