Animal DNA-dependent RNA polymerases 11. Mechanism of the inhibition of RNA polymerases B by amatoxins

The mechanism of the inhibition of purified calf thymus RNA polymerases B by amatoxins has been studied. Although binding of the enzyme to DNA is not inhibited, initiation of RNA synthesis as well as PPi exchange appear to be inhibited to the same extent as chain elongation. Amatoxins do not affect the affinity of RNA polymerases B for any of the four nucleoside triphosphates. There is no release of DNA or RNA when amatoxins are added to a ternary enzyme—DNA—RNA complex. It appears therefore that amatoxins act primarily by preventing the formation of phosphodiester bonds. A sensitive membrane filter assay for amatoxins—RNA polymerases B complex has been used for equilibrium and kinetic studies of amatoxins and polymerases B interaction. It was demonstrated that the binding of one amatoxin molecule per enzyme B molecule is responsible for the observed inhibition of RNA synthesis. The equilibrium association constant (KA) is high, in the order of 108–1010 M−1, depending on the temperature and on the ionic strength of the reaction. The rate of dissociation (k2) of the complex, which follows first-order kinetics, is slow and is markedly decreased when the temperature is decreased. A decrease of the rate of dissociation is also observed when the ionic strength is increased. The rate of association (k1) of amatoxins and enzymes B follows second-order kinetics and is much less sensitive to change in temperature than the rate of dissociation. The ratio k1k2 has been found to agree well with the association equilibrium constant KA over a wide range of temperature, except around 0°C. The driving force for the binding reaction comes from a variation of enthalpy of about −20 kcal · mole−1, whereas the entropy change is about −26 cal · mole−1 · degree−1. The results indicate that electrostatic forces are not important in enzymes B—amatoxin interaction. Hydrogen bonds seem to play an essential role in the stability of amatoxin—enzymes B complex as suggested by the values of the thermodynamic parameters and by the study of the dissociation rate constant of complexes between enzymes B and various amatoxins, differing only in the structure of their hydroxylated isoleucine side-chain. However, the overall conformation of amatoxins is of primer importance for their ability to bind and to inhibit RNA polymerases B. There is a good correlation between the affinity of the different amatoxins for calf thymus RNA polymerases B and their in vivo toxicity. Comparison of the association and dissociation rate constants of α-amanitin and O-methyl-demethyl-γ-amanitin—enzymes B complexes with their in vivo toxicity suggests that the factor which governs the in vivo toxicity is not the association rate, but the dissociation rate of the complex.