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.

Inductive effects on the structure of proline residues.

4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)- fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C gamma-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1,2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.

NMR strategy for determining Xaa-Pro peptide bond configurations in proteins: mutants of staphylococcal nuclease with altered configuration at proline-117.

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro117 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the H alpha of residue Xaa and the proline H delta or H alpha protons. Short (< 2.5 A) Xaa H alpha-Pro H delta interproton distances are diagnostic for the trans configuration, whereas short (< 2.5 A) Xaa H alpha-Pro H alpha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-13C]Xaa and [delta-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-13C]Xaa and [alpha-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off-diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the Lys116-Pro117 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine 1H epsilon 1 NMR signals.(ABSTRACT TRUNCATED AT 250 WORDS)