Value of general Acid-base catalysis to ribonuclease a.

Bovine pancreatic ribonuclease A (RNase A; EC 3.1.27.5) has been one of the most studied of all enzymes.’ RNase A efficiently catalyzes the cleavage of RNA.2 Early X-ray diffraction analyses revealed that the active site of RNase A contains two histidine residues, histidine 12 (H12) and histidine 119 (H1 19).3 The results of chemical modification4 and pHrate’ studies are consistent with an enzymatic reaction mechanism in which the rate-limiting transition state for RNA cleavage is similar to that shown in Figure 1. In this mechanism, theimidazole side chain of H12 acts as a general base by deprotonating the 2’ oxygen, and that of H119 acts as a general acid by protonating the 5” oxygen. These two residues have evoked much interest in bioorganic chemistry: as well as in protein chemistry and enzymology.’ Indeed, no residue other than H12 and H119 need be invoked to explain the classic bell shape of the pH-rate profile’ for catalysis by this enzyme. Here, we report the explicit value of this general acid and this general base to catalysis of RNA cleavage by RNase A. We used recombinant DNA techniques to produce mutant ribonucleases in which either H12 or H119 was changed to an alanine residue.’ This change effectively substitutes a proton for the imidazole group of each residue. We then determined the ability of the resulting mutant enzymes, H12A RNase A and H119A RNase A, to catalyze thecleavageof three phosphodiester substrates: polycytidylic acid [poly(C)], uridylyl(3’+5’)-