Zum Mechanismus der Ribonuclease‐Reaktion

Abstract

The hydrolysis of cyclic 2′,3′‐cytidylic acid (Cp) and cyclic 2′,3′‐uridylic acid (Up) by panereatic ribonuclease (E.C.2.7.7.16) at pH 7 and 5.6 is activated by adenine, adenosine, cyclic 2′,3′‐adenylic acid, and adenylyl‐adenosine (ApA). The activation is reflected by parallel shifts in the kinetic parameters when expressed on a e/v —1/ s ‐diagram. The measured parameters k° =π· k +2 and K° =Φ· K m are increased by the same factor, i.e. Φ=π > 1 or 1 . Rate equations can be derived for an interaction of the modifier with either the substrate, the enzyme, or the enzyme‐substrate‐complex (ES), independent of any mechanistic concept. It is then possible to calculate the conditions pertaining to the relation Φ=π. The first case, an interaction between the modifier and the substrate can be excluded. In the case of an interaction between the modifier and the enzyme or ES the rate equations if Φ=π require that k −1 and k +2 are equally influenced by the modifier. Thus both constants are coupled and associated with reactions of the same type. Since the dissociation of the inhibitor 3′‐Cp from the enzyme as measured by relaxation times [21] is 10 3 times faster than the k +2 value in the reaction where the same 3′‐Cp is formed as the product, k +2 cannot be the dissociation of the product from the enzyme, but must be associated with a step involving bond exchange. Consequently k −1 must be associated with a step which also involves a bond exchange and is catalysed by the same principle. Both steps should therefore start from a common intermediate and lead alternatively either to the substrate ( k −1 ) or to the product ( k +2 ). It can be further shown that k +1 must be associated with the complementary step to k −1 , also involving bond exchange, and that a simple association‐dissociation‐equilibrium between enzyme and substrate (not involving covalent bond exchanges in the substrate) does not appear in the measurable kinetic parameters. K m therefore would not represent a dissociation constant in the sense of Michaelis‐Menten but rather the ratio of the rate constants for the breakdown and the formation of an intermediate which is stabilized by the enzyme. The mathematical relations do not give any information as to the nature of the bonds exchanged or the type of catalysis. On the basis of the mechanism derived from other experiments in our previous investigations [1,3] it can be concluded now that the enzyme only induces a non‐rate‐limiting polarisation at the monoanionic phosphodiester group of the substrates. Binding would occur in a very fast non‐rate‐limiting step after the rate limiting base catalysed step when the dianionic intermediate has been formed.