On the mechanism of the ribonuclease reaction. 2. The pre-ordering in the substrate as the accelerating factor in cinucleoside phosphates and analagous compounds

Abstract

In the previous paper [1] we proposed a mechanism for the reaction of pancreatic ribo‐nuclease with nucleotide diesters, in which the kinetic constants k+1 and k−1+k+2 are assigned to a base eatalysed step for the formation, and a proton catalysed step by the conjugate acid for the breakdown, of an enzyme stabilized intermediate. In the hydrolysis reaction of the 2′,3′‐cyclic diesters the rates depend on the structure of the catalysing base, and in particular on its polarisability. In addition to this effect, we found that, in the dinucleoside phosphate series, the rates increased up to three orders of magnitude, depending on the nature of the base of the second nucleoside [4].According to our suggested mechanism I–VI the accelerated rates could be due to (a) the second base influencing the polarisability of the catalysing system by π‐electron interactions between the stacked bases. This effect should be reflected in the enthalpy term and should parallel the hypochromicity of the dinucleoside‐phosphates exactly; (b) pre‐ordering of the reacting atoms (C‐2‐oxygen, 2′‐OH‐group and phosphorus) by the base stacking, which would be reflected in a lowering of the activation energy in the entropy term and should only roughly parallel the hypochromicity.To differentiate between these two possibilities we prepared the diesters IX–XXXIV, determined the hypochromic effect and also the values of Km and k+2 at pH 7. There is only a partial correlation between k+2 values and the hypochromic effect. The k+2 values vary with the structure of the second nucleoside and show a particular dependence upon the nature of the link between base and phosphate group. Highest values are found with adenosine and deoxy‐adenosine. Elongating the ribose with mercaptoethanol (XIX) or substituting the relatively rigid ribofuranose ring with flexible n‐hexyl‐, pentyl‐ or butyl‐chains (XX– XXII) appreciably reduces the hypochromicities and also the k+2 values. In the case of a propyl link (XXIII, XXIV) or in the analogously structured 3′‐3′‐dinucleosidephosphate XXV no hypochromic effect is found and the k+2 values are as low as that of 3′‐cytidylic acid benzyl ester.A second nucleoside attached to the 5′ end of a substrate (XXXIII, XXXIV) does not result in an increase of the rate of diester hydrolysis at the 3′ end though there is a normal hypochromicity. Therefore a pre‐ordering of the reacting atoms (demonstrable on models) appears to be the accelerating factor. A specific interaction between the second nucleoside and the enzyme being responsible for the pre‐ordering effect can be excluded by either blocking or varying all the potential interaction sites.The concept of a pre‐ordering in the substrate due to base stacking agrees with the results from measurements of the temperature dependence of k+2 and calculations of the enthalpy (ΔH≠) and entropy (ΔS≠) term in the activation energy of this step. When diesters with an exactly analogous structure of the intermediate II such as CpA (IX) and Cp‐butyladenine (XXII) are compared, the differences are found in the entropy term only. Between the first and second step substrates such as CpA and 2′,3′‐Cp the differences are also found in the entropy term with a small factor in the enthalpy term.From a comparison of Up‐ and Cp‐diesters it can be seen that due to the differences in the enthalpy term the Up‐diesters should react 5–10 times faster than Cp‐diesters, but that due to the differences in the entropy term Cp‐diesters react 20 times faster than Up‐diesters. This is in agreement with our concept that the enthalpy term of the activation energy is derived from the transfer of a proton from the conjugate acid of the pyrimidine base to the 5′‐ (or 2′‐) oxygen, what should be easier the more acidic the conjugate acid is. However, the probability that the base is in the form of its conjugate acid and in the right position to transfer the proton is appreciably higher in the case of the more basic cytidine than with uridine. This explains the observations made in the previous paper [1] that, in spite of an acid‐base‐catalysis mechanism for k+1 and for k−1 and k+2, the rates do not directly parallel the acidity or basicity of the catalysing groups.