CONTRIBUTION OF A TYROSINE SIDE-CHAIN TO RIBONUCLEASE-A CATALYSIS ANDSTABILITY

An intricate architecture of covalent bonds and noncovalent interactio ns appear to position the side chain of Lys 41 properly within the act ive site of bovine pancreatic ribonuclease A (RNase A). One of these i nteractions arises from Tyr 97, which is conserved in all 41 RNase A h omologues of known sequence. Tyr 97 has a solvent-inaccessible side ch ain that donates a hydrogen bond to the main-chain oxygen of Lys 41. H ere, the role of Tyr 97 was examined by replacing Tyr 97 with a phenyl alanine, alanine, or glycine residue. All three mutant proteins have d iminished catalytic activity, with the value of k(cat) being perturbed more significantly than that of K-m. The free energies with which Y97 F, Y97A, and Y97G RNase A bind to the rate-limiting transition state d uring the cleavage of poly(cytidylic 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 c atalysis. The role of Tyr 97 in the thermal stability of RNase A is la rge. The conformational free energies of native Y97F, Y97A, and Y97G R Nase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. T he unusually large decrease in stability caused by the Tyr --> Phe mut ation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.