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 examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during 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 catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 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.
Eberhardt
E S
ES
Department of Biochemistry, University of Wisconsin-Madison 53706-1569, USA.
Wittmayer
P K
PK
Templer
B M
BM
Raines
R T
RT
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
D011994:Recombinant Proteins; D014443:Tyrosine; D012259:Ribonuclease, Pancreatic