The special characteristics of the enzymes that constitute the ribonuclease superfamily make them ideal candidates for the study of mechanisms related to protein folding and enzyme catalysis. Apart from this, some members of this superfamily show a very interesting biological activity which could eventually be used pharmacologically. In this project, anc with the use of techniques of site-directed mutagenesis, we will try to get a deeper understanding of the following aspects: 1) an assessment of the contribution of certain amino acids to catalysis and specificity of the enzymic reaction. In particular, the formation of an additional catalytic site after substitution of the amino acids involved in the p\sub 2\nosub subsite by histidine. The unambiguous characterization of this new site implies doing strict controls in which the histidines of the original native catalytic site will be surpressed. We shall also try define the factors affecting the change in specifity (specially the change from endonuclease to exonucleases activity) found when polynucleotides are used as substrates. 2) Studies on the nucleation core at the C-terminus which allows folding of ribonuclease A to the native conformation will be carried out; special focus will be put on the degree of hydrophobicity of certain amino acids. The whole process will be compared with that for human pancreatic ribonuclease which contains three additional prolines. As the presence of prolines gives rise to the appearance of cis-trans isomerism and as the transformation between isomers is a slow process, modification of these additional prolines should result in new insight on the rate-limiting step of folding. 3) We shall try to find which parts of the ECP (eosinophile cationic protein) molecule, which shows citotoxic and enzymic activities, are responsible for each of these activities. 4) Finally, the interaction between human pancreatic ribonuclease and the protein ribonuclease inhibitor will be studie
|Effective start/end date||1/12/97 → 1/12/00|
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.