This project is concerned with obtaining detailed structure-function correlations for members of the ribonuclease superfamily. This family of proteins includes digestive enzymes such bovine pancreatic ribonuclease (RNase A), toxins, such as human eosinophil derived neurotoxin, growth factors such as human angiogenin, and P-30, a protein with significant toxicity against human tumor cell lines. The sequence homology and ribonucleolytic activity of all these proteins, suggests that they share a common catalytic mechanism and tertiary structure. The vast amount of structural and biochemical information available for RNase A provides a unique opportunity to examine the basis for the biological activities displayed by members of the ribonuclease superfamily. RNase A will be systematically mutated towards the primary sequence of these homologs, and the hybrid proteins will be examined by X-ray crystallography, kinetics and the appropriate bioassays. In every case, the biological activities have been linked to the ribonucleolytic activity. It is therefore crucial to understand the detailed enzymatic mechanism(s) used by the various homologs. In addition to unraveling the connection between enzymatic activity and biological function, the production of hybrid proteins will allow for the study of phenomena which are of fundamental importance to enzymology. Specifically, members of the ribonuclease superfamily utilize the binding energy of extended substrates to enhance catalytic efficiency by up to four orders of magnitude. Furthermore, the rate of catalysis in the superfamily is highly sensitive to the identity of amino acids which are not directly involved in either the chemical transformation or substrate binding: kcat/Km spans six orders of magnitude across the superfamily. How enzymatic activity is effected by interactions distant from the site of chemistry is one of the outstanding questions in enzymology, and is of general importance. The production of hybrid ribonucleases will allow for an examination of important biological activities such as neurotoxicity, growth factor activity and tumor cytotoxicity. In addition, these same studies will address the fundamental question of 'long range distant effects' in catalysis. Defining the structural and chemical determinants responsible for these biological activities will be instrumental in understanding their underlying mechanism and in providing a basis for therapeutic development.
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