Zum Mechanismus der Ribonuclease‐Reaktion

The 2′,3′‐cyclic phosphates VII–XXXI were prepared for further studies of the function of the base in the ribonuclease‐catalysed hydrolysis of ribonucleotide diesters. As seen with purine (XXIV, XXV, XXVIII), pyridine (XXIX), and pyridazine (XXXI) derivatives there is no absolute specificity for pyrimidine bases. Minimal requirement for monomeric substrates appears to be a mesomeric ring system with a keto‐group in α‐position to the β‐glycosidic bond. Specific binding to the enzyme by position 3, 4, 5, or 6 of the base can be excluded. Arguments that the keto‐group is not involved in the binding process are derived from kinetic experiments. Alterations at the base do not affect K m but only k +2 . A relation between structural alterations at the substrates and the measured kinetic parameters k +2 and K m = ( k −1 + k +2 )/ k +1 , required an analysis of the significance of these values in connection with definite reaction steps. This is accomplished on the basis of the mechanism I–VI [3] in which the reaction occurs by a simultaneous bond exchange at three centers (see XXXV–XXXIV) initiated by a base catalysis with the 2‐oxygen of the base and a polarisation at the phosphate group by two proton donating groups of the enzyme [4]. Binding to the enzyme occurs only when the dianionic intermediate state II or V has been formed. This complex breaks down after a proton catalysis by the conjugate acid of the base, which can occur alternatively in two directions indicated by k −1 and k +2 . Arguments are given that rate determining in k +1 is the transfer of the proton of the 2′‐OH group to the 2‐oxygen of the base in I → II or by analogy in IV → V. Rate determining in k −1 and k +2 is the proton transfer from the conjugate acid to the 2′‐oxygen in II → I and to the 5′‐oxygen or its equivalent in II → III and by analogy in V → IV and V → VI. Both constants are coupled as shown in [32] and change by the same factor, when the catalytic function of the base is altered. If in such a case K m remains constant, k +1 must have changed by the same factor too. Thus K m cannot be treated as K s = k −1 / k +1 with k +2 negligibly small compared with k −1 . Further result [31] indicate that the activation energy for k +1 contains an enthalpy term which is related to the transition XXXVII → XXXVIII and an entropy term, related to the probability of the base being in the proper position to accept the proton. Similarly k −1 and k +2 contain an enthalpy term related to the transition XXXVIII → XXXVII and an entropy term related to the probability of the base being in the form of the conjugate acid and in the proper position to transfer the proton. Our findings that the rates depend on the polarisability of the base with K m remaining constant and not on the acidity or basicity of the catalysing base can be explained by the assumption that increased polarisability lowers the activation energies for the transitions XXXVII ⇄ XXXVIII in both directions thus changing k +1 and k −1 + k +2 by the same factor. Increased acidity, however, would lower the activation energy for k +2 in the enthalpy term, but at the same time would increase it in the entropy term; it is less probable to have the less basic base in the form of the conjugate acid. The higher rates which we found when the conjugate acid can be formed by a tautomerisation of the catalysing base can be similarly explained. Further factors influencing the rates are of sterical nature. We found differences when the catalysing α‐oxygen is part of a five‐membered or of a six‐membered ring system or when the catalysing oxygen cannot offer its free orbital to the 2′‐OH‐group as discussed in the case of α‐cytidylic acid (XXIII). Another factor, the pH‐dependence of k +2 , will be discussed in a further paper [23] on the basis of an additional catalysis of the decay by an enzyme base with a pK around 6.5.