ABSTRACT
A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters has been carried out for onconase. Two main hydrophobic nuclei in the onconase structure were detected. Their composition and shape were found to be very similar to those of RNase A, in accordance with the predictions made. The nuclei in onconase are more compact, the side-chain atoms of residues included in the nuclei in onconase form more contacts with the environment than in RNase A. The hydrophobic nuclei should be considered as individual structural units along with elements of the secondary structure. Differences in composition and conformation of exposed loops between onconase and RNase A were found. The additional hydrophobic clusters attached to the nuclei in onconase might be involved in the fixation of an appropriate conformation of site(s) for manifestation of the biological activity of onconase. A comparison of amphibian representatives of the RNase A superfamily was also made. The results obtained suggest that the availability of nonpolar residues in established key positions of amino acid sequences determines the characteristic fold of homologous proteins and the structure of the active site cleft.
Subject(s)
Egg Proteins/chemistry , Egg Proteins/metabolism , Ribonucleases/chemistry , Ribonucleases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Folding , Protein Structure, Secondary , Ranidae , Ribonuclease, Pancreatic/chemistry , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis. Mutation of His119 to asparagine was generated to study the role of His119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His119 of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His119 participates directly in a transition state stabilization via hydrogen bonding.
