碩士 / 國立臺灣大學 / 生物化學暨分子生物學研究所 / 94 / Shrimp hepatopancreatic nuclease（shNuclease）, previously designated as DNase I, has RNase activity in the presence of Mg2+ and Ca2+ ions. The shNuclease precusor contains 21-residue signal peptide and a 381-residue mature protein. The enzyme has 11 cys residues, forming five intramolecular disulfide bonds.
Sequence alignment revealed there is no homologous protein. However, residues 205-255 shared a conserved active-site motif within a distinct group of nucleases, including Serratia marcescens. According to the previous report, Arg57、Arg87、His89、Asn119 and Glu127 of the nuclease of Serratia marcescens（Serratia nuclease）are very important in catalysis. And the five critical residues of Serratia nuclease are equivalent to Asn179、Lys209、His211、Asn241 and Glu249 of shNuclease. His211 showed to be in the centre of active site.
In our previous studies, the recombinant shNuclease expression system was established and the K209R、K209L、H211Q、E249D and E249Q variants were constructed. The expressed proteins showed that the specific activities of K209R increased 1.7-fold as that of wild type shNuclease, while those of other variants decreased drastically, indicating His211 and Glu249 could be directly involved in catalysis. Lys209 could be the substrate binding residue in shNuclease corresponding to Arg87 in Serratia nuclease. Because Arg has stronger basicity than Lys, replacing Lys209 with Arg may promote the substrate binding ability of shNuclease. Therefore the DNase activity of K209R in shNuclease was increased [33].
To further investigate the catalytic mechanism, we constructed N179A, N179R, N179D, K209A, N241D and N241Q variants. Wild type and mutated recombinant shNucleases were expressed in human 293T cell. The recombinant shNuclease in the culture media was collected and purified with Ni-NTA agarose beads. Based on the intensities on Western blots and zymograms, the specific activity of N179A, N179R and N179D variants were 0.7-fold, 1.58-fold and 0.59-fold as compared with that of wild type shNuclease, respectively. Friedhoff et. al proposed that Arg57 of Serratia nuclease participated in transition state stabilization. Since Arg57 of Serratia nuclease is equivalent to Asn179 of shNuclease, we suggested that Asn179 might play the same role. When replacing Asn179 with Arg, the positive charge of Arg may promote the transition state stabilization, therefore the DNase activity of N179R shNuclease was increase. The negative charge of Asp may reduce the DNase activity of shNuclease more than the uncharged Ala residue. Because K209A had no DNase activity, we suggest that Lys209, which is positivily charged, may bind DNA in shNuclease. Because N241D and N241Q had no DNase activities, we suggested that Asn241 might participate in catalysis. Like Asn119 in Serratia nuclease, Asn241 might participate in the transition state stabilization or involed in ligand binding of the essential cofactor, Mg2+. Our results were similar to that of Friedhoff et. al. Therefore we proposed that the catalytic mechanism of shNuclease might be similar to Serratia nuclease.
| Identifer | oai:union.ndltd.org:TW/094NTU05104004 |
| Date | January 2006 |
| Creators | Hsiao-Chieh Fan, 范筱婕 |
| Contributors | 廖大修 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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