Ribofuranose Ring Research Articles

The conformational analysis of adenosine triphosphate by classical potential energy calculations

AbstractThe conformational analysis of adenosine triphosphate was conducted by using classical potential energy calculations. All rotatable bonds were examined, i.e., no dihedral angles were fixed at predetermined conformations except for the ribofuranose ring, which was held in the C(3′)‐endo conformation—the conformation observed for adenosine in the crystal state.The energy terms included in the total energy expression consist of nonbonded pairwise interaction, electrostatic pairwise interaction, free energy of solvation, and torsional bond potentials.Two separate approaches were used in the conformational analyses. The first consisted of a sequential fragment approach were four bonds were rotated simultaneously at 30° increments. Each fragment overlapped the preceding one by at least one bond. All rotors were then simultaneously examined at their minima and at ±15°. The second approach consisted of a coarse grid search where all rotors were examined simultaneously, but only at staggered positions. The low‐energy conformations thus obtained were then used as starting conformations for a minimization routine based on the method of conjugate directions. The first approach required about 40 hr of central processing unit (CPU) computer time, while the coarse grid/minimization approach required about 4 hr of CPU time.Both the sequential fragment approach and the minimization approach yielded lowest‐energy conformations which are remarkably similar to the solid‐state conformation of C(3′)‐endo ATP.

Studies of the Binding of Phosphoribosyl Pyrophosphate to Adenine Phosphoribosyltransferase

Abstract The groups on magnesium phosphoribosyl pyrophosphate and on adenine phosphoribosyltransferase which are involved in their binding were investigated by comparing the inhibition constant of magnesium phosphoribosyl pyrophosphate with those of its analogues, by determining the effect of pH on the Michaelis constant of this substrate, and by protection by substrate and substrate analogues against enzyme inactivation by p-hydroxymercuribenzoate and other reagents. The 1-magnesium pyrophosphoryl and 5-phosphoryl moieties are the most important points of binding, and the main role of the ribofuranose ring seems to be to keep these anionic groups at a critical distance. The enzyme is inhibited by p-hydroxymercuribenzoate, but this is prevented by magnesium phosphoribosyl pyrophosphate. A group on the enzyme which has an ionization constant of 9.35 is involved in binding of this substrate.

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

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.