RESUMEN
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.
Asunto(s)
Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Dimerización , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Isomerismo , Cinética , Concentración Osmolar , Poli A/genética , Poli T/genética , Estructura Cuaternaria de Proteína/efectos de los fármacos , Subunidades de Proteína , Cloruro de Sodio/farmacología , TermodinámicaRESUMEN
The synthesis of new 1,3-phenylene derivatives and their preliminary evaluation as antivirals (Herpes simplex 1, HSV-1) whose antiherpetic activity can be related with the inhibition of the interaction of the origin binding protein (OBP) with the DNA are presented. The new compounds are adjusted to a previously defined common structural model, consisting of a central aromatic system, which presents two side chains of different lengths in relative position 1, 3; these chains are made up of atomic groups characterized by the alternation of positive and negative centers, situating differently substituted rings, preferably aromatic, at the ends of both chains. Some of these derivatives, such as N,N''-(4-methoxy-1,3-phenylene)bis[N'-(4-nitrophenyl)urea] (2c) or (1,3-phenylene)bis[N-(p-tolyl)aminosulfonyl] (11b), show antiherpetic activity related to the proposed mechanism.