核醣核酸水解酵素普遍存在於自然界中，而且對核醣核酸的代謝非常重要。近年來科學家發現屬於牛胰臟核醣核酸水解酵素（RNase A）群的酵素，不但具有酵素活性而且具有許多其他的生化功能。我們實驗室自牛蛙中也純化出8種具有細胞毒性的核醣核酸水解酵素，由序列分析顯示他們也都是屬於RNase A核醣核酸水解酵素群的成員。為了瞭解這些蛙類核醣核酸水解酵素和其他RNase A核醣核酸水解酵素群成員間的異同點，首先我們分析這些酵素的特性。結果發現這些蛙卵中的核醣核酸水解酵素胺基酸序列相似度都很高，他們的酵素比活性差距很大、最佳反性酸鹼值分佈很廣、專一性不一、細胞毒性也不相同。但是反應過程都不需要離子的參與，對熱的穩定性也都高於RNase A。接著我們進一步分離這些核醣核酸水解酵素的基因，並且利用點突變、大腸桿菌製造和管柱層析法純化的技術得到突變蛋白，分別測試突變蛋白酵素活性、專一性和動力學的變化。另外依據牛蛙RC-RNase和受質類似物的晶體結構，我們得以了解RC-RNas與受質間的作用機制，結果發現RC-RNase的Pyr1參與酵素活性及專一性；Lys9、His10、Lys35和His103參與P1活性區；Thr39、Thr70和Phe104參與B1受質結合區，其中Thr39直接與受質結合，Thr70透過Thr39影響受質專一性，而Phe104與受質為疏水性作用。Lys95和Glu97參與B2受質結合區，其中Lys95與受質結合，而Glu97不但與受質結合，同時也會固定Lys95支鏈的位置。由這些結果我們得以進一步了解這些蛙類核醣核酸水解酵素的作用機制。

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-RNase□d(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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