5 X10^-9 M.

In a very detailed study, Cochet-Meilhac & Chambon (1974) demonstrated that the amatoxins bind to RNA polymerase B with a 1:1 stoichiometry, that the affinity of the toxin for the enzyme is very strong with an equilibrium association constant of 3.5 X 10^-8 M^-1 at 30 ℃, and that the dissociation is very slow and highly dependent upon temperature and ionic strength. Subsequently, it was shown by Brodner & Wieland (1976), using a carbodiimidecondensation reaction of labeled amatoxin to calf thymus RNA polymerase B, that the 140-kilodalton subunit was a binding site for the amatoxins. Thus, it has been clearly shown that inhibition by a-amanitin is the result of a direct interaction of the toxin with RNA polymerase B.

The process of RNA synthesis catalyzed by DNA-dependent RNA polymerase is quite complex and may be described in several steps as follows: (a) formation of a stable binary complex with the DNA template; (b) RNA chain initiation; (c) translocation and elongation of the nascent RNA; (d) termination and dissociation of the RNA chain. Hence, there are a number of enzyme mechanisms which could possibly be specifically disrupted by a-amanitin. It has been shown that the binding of a-amanitin does not cause a dissociation of the DNA-RNA polymerase B binary complex or the dissociation of the DNA-enzymeRNA ternary complex (Cochet-Meilhac & Chambon, 1974). In the same study, direct evidence was given that the binding of a-amanitin was responsible for the inhibition of RNA chain elongation as catalyzed by RNA polymerase B. The inhibition of chain initiation was suggestedn by indirect experimentation, i.e., by a failure to detect pyrophosphate exchange which is presumably the reverse reaction of phosphodiester bond formation (Krakow & Fronk, 1969). Therefore, it was concluded by Cochet-Meilhac & Chambon (1974) that the binding of amatoxin to RNA polymerase B prevents the formation of phosphodiester bonds.

Reference:

Vaisius, A. C., & Wieland, T. (1982). Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by. alpha.-amanitin. Biochemistry, 21(13), 3097-3101.