Factors affecting the dimerization of the p66 form of HIV-1 reverse transcriptase.
The association and dissociation of the homodimeric p66/p66 form of HIV-1 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 size-exclusion 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 1 M decreased the equilibrium dissociation constant from 2.0 to 0.34 microM. Analysis of the kinetics of the dimerization process indicated it followed a two-step 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)/dT(20) decreased the equilibrium dissociation constant of the homodimer about 30-fold, while the addition of 5 microM 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 low-ionic-strength buffer. The results suggest that binding of immature HIV-1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.
Department of Biochemistry and Molecular Biology, University of Navarra, Pamplona, Spain.
J F
JF
Cabodevilla
L
L
Odriozola
E
E
Santiago
J J
JJ
Martínez-Irujo
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D009994:Osmolar Concentration; D011061:Poly A / Q000235:genetics; D011071:Poly T / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021122:Protein Subunits; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics
D021122:Protein Subunits; D011061:Poly A; D011071:Poly T; C008950:poly A-T; D012965:Sodium Chloride; D054303:HIV Reverse Transcriptase