Mechanistic characterization of the HDV genomic ribozyme: Assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism

Hepatitis delta virus (HDV) uses genomic and antizgenomic ribozymes in its replication cycle. We examined ribozyme self-cleavage over eight orders of magnitude of Mg2+ concentration, from approximate to 10(-9) to 10(-1) M. Th ese experiments were carried out in 1 M NaCl to aid folding of the ribozyme and to control the ionic strength. The concentration of free Mg2+ ions was established using an EDTA-Mg2+ buffered system. Over the pH range of 5-9, the rate was independent of Mg2+ concentration up to 10-1 M, and of the add ition of a large excess of EDTA. This suggests that in the presence of I M NaCl, the ribozyme can fold and cleave without using divalent metal ions. B ronsted analysis under these reaction conditions suggests that solvent and hydroxide ions may play important roles as general base and specific base c atalysts. The observed rate constant displayed a log-linear dependence on i ntermediate Mg2+ concentration from approximate to 10(-7) to 10(-4) M. Thes e data combined with the shape of the pH profile under these conditions are consistent with the binding of at least one structural divalent metal ion that does not participate in catalysis and binds tighter at lower pH. No ev idence for a catalytic role for Mg2+ was found at low or intermediate Mg2concentrations. Addition of Mg2+ to physiological and higher concentrations , from 10-1 to 10-1 M, revealed a second saturable divalent metal ion which binds tighter at high pH. The shape of the pH profile is inverted relative to that at low Mg2+ concentrations. consistent with a general acid-base ca talysis mechanism in which a cytosine (C75) acts as the general acid and a hydroxide ion from the divalent metal ion, or possibly from solvent, acts a s the base. Overall, the data support a model in which the HDV ribozyme can self-cleave by multiple divalent ion-independent and -dependent channels., and in which the contribution of Mg2+ to catalysis is modest at approximat ely 25-fold. Surface electrostatic potential maps were calculated on the se lf-cleaved form of the ribozyme using the nonlinear Poisson- Boltzmann equa tion. These calculations revealed several patches of high negative potentia l, one of which is present in a cleft near N4 of C75. These calculations su ggest that distinct catalytic and structural metal ion sites exist on the r ibozyme, and that the negative potential at the active site may help shift the pK(a) for N3 of C75 toward neutrality.