The Roles of Histidine Residues in Deoxyribonuclease II

• Main Authors: Yu-Che Cheng, 鄭宇哲
• Other Authors: Ta-Hisu, Liao
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/80556254896637783809

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 94 === Deoxyribonuclease II (DNase II) is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different His-to-Leu mutants of porcine DNase II, and examined the enzyme properties of the mutant proteins. Of the nine mutant proteins, all but H132L were secreted into the growth media of H293Tcells. Six of the mutated DNase II proteins showed enzymatic activities (H41L, H109L, H206L, H207L, H274L and H322L with specific activities of 223.2, 286.9, 218.8, 135.0, 178.2, 320.0 U/mg, respectively), whereas the H115L, H132L and H297L mutant proteins exhibited very little activity. The H115L and H297L mutant proteins were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II; these mutant proteins were inactive, but their DNase activities could be rescued with imidazole. Addition of 100 mM imidazole increased the DNase II activity of H115A by approximately 7-fold and that of H297A by approximately 11-fold. We next assessed the chemical rescue of H115A and H297A in buffers with different pH values. The pH-activity profiles showed the optimum pH for the imidazole-based restoration of catalytic activity was more acidic for H115A versus H297A, indicating that H115 and H297 are likely to function as a general acid and a general base, respectively, in the catalytic center of the enzyme. In contrast to the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for H132 with Lys resulted in the misfolded form and retained in endoplasmic reticulum. In conclusion, we identified the H115 is a general acid whereas H297 is a general base in the catalysis of DNase II. H132 is a key residue for DNase II folding, other His residues are dispensable. The results provide important new insights into the catalytic mechanism and protein folding in DNase II.