Contribution of a tyrosine side chain to ribonuclease A catalysis and stability

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was exam-ined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have dimin-ished catalytic activity, with the value of kc,, being perturbed more significantly than that of K,,,. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidy1ic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal sta-bility of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are de-creased by 3.54, 12.0, and l l.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr + Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond