Studies of models for ribonuclease catalysis

The aim of this research has been to gather some insight into the mechanism of hydrolysis of ribonucleic acid, now known to take place in two distinct stages under catalysis by enzyme ribonuclease A. The relatively fast first step of the hydrolytic sequence is the intramolecular transesterification reaction which give rise to a series of intermediate nucleotides containing five-membered phosphate diester rings. This is followed by a relatively slow hydrolysis of the cyclic nucleotides in which the ring-opei.ing of the intermediate produce the final nucleotide products. In order to study the rate of the sequence, an analogous model compound, methyl cis-3-hydroxytetrahydrofuranyl-4-phosphate was studied in detail. The model compound for the ring-opening reaction in the second step of the sequence was cis-3,4-tetrahydrofurandiol phosphate. It has been found that the ring-closure reaction of the g-hydroxydiester model compound is subject to much more pronounced positive salt effect than the subsequent ring opening reaction when the reactions are studied in aqueous base. However, the rate of the ring-closure step of the hydrolytic sequence increased significantly in alkaline aqueous DMSO, the subsequent ring-opening reaction under identical experimental condition did reveal little or no rate enhancement. Measurement of activation parameters did indeed reveal that the base-catalyzed ring-closure step of the model studies in DMSO is accompanied by about 1.7 Kcal/mole favorable enthalpy advantage compared to that in plain aqueous medium. This is believed to be the result of enhanced ionization of the g-hydroxyl proton in alkaline DMSO. Another striking result has been a 2-3 fold rate enhancement observed for the rate of base-catalyzed ring-closure step in cationic micelle CTABr. Corresponding reaction for the ring-opening step has been found to be subject to slight inhibition by added detergent CTABr. These results have been rationalized by assuming that the B-hydroxyphosphate diester has different requirement for hydrophobic and electrostatic interaction with the micellar phase in the ground state and these interactions are favorable for the ring-closure step under the conditions of the experiment. Without the necessary activation parameters, it is not possible to say whether the lowering of the free energy of activation is the consequence of favorable activation enthalpy or entropy or both. The acid-catalyzed ring-closure and ring-opening reactions of the model compound did indicate that the effect of the tetrahydrofuranyl ring is very large on the acid-dissociation constant of the B-hydroxy phosphate diester as well as the cyclic one. No departure from second-order dependency on the concentration of the acid was observed in the pH range 0.72-1.26 for either steps of the hydrolytic sequence with the model compound. An anionic detergent NaLS was found to have no measurable effect on the rate of either step of the hydrolytic sequence.