| Summary: | 博士 === 國立清華大學 === 生命科學系 === 91 === Ribonucleases are widely found in prokaryotes and eukaryotes and play an important role in RNA metabolism. Several ribonucleases in the RNase A superfamily not only possess ribonucleolytic activity but also exhibit other functions. In our laboratory, eight different ribonucleases were isolated from Rana catesbeiana (bullfrog). They show sequence homology to the RNase A superfamily. A series of biochemical and biophysical analyses have been exploited to characterize these novel ribonucleases. Although these ribonucleases have some characteristics in common, such as high thermo stability and lack of ions in their enzymatic reaction, they are different in catalytic activities, optimal pH values for enzyme activities, substrate specificities and cytotoxicities. To further investigate the mechanisms of substrate recognition, their cDNAs were cloned. Recombinant wild-type and mutant proteins were produced in Escherichia coli and subjected to analyses of enzyme activity, substract specificity and kinetics. Cocrystalization of RC-RNased(ACGA) further revealed the mechanism of substrate specificity determination in more detail. Pyr1 is involved in both enzyme activity and substrate specificity. The residues of Lys9, His10, Lys35 and His103 contribute to the P1 active site. RC-RNase contains two base binding pockets, B1 and B2. In the B1 site, Thr39 directly binds to the substrate, whereas Thr70 influences the substrate specificity through Thr39 and Phe104 works through the hydrophobic reaction with the substrates. In the B2 site, both Lys95 and Glu97 bind to the substrate and Glu97 also supports the side chain of Lys95. With these results, we established the relationship between ribonucleases and their substrates for further investigation.
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