- 作者: Shih-Lu Wu; Chia-Cheng Li; Jaw-Chyun Chen; Yi-Jin Chen; Ching-Ting Lin; Tin-Yun Ho; Chien-Yun Hsiang
- 作者服務機構: Department of Biochemistry, China Medical University, Taichung, Taiwan, R.O.C.
- 中文摘要: --
- 英文摘要:
Background: Endonuclease G (EndoG), a member of DNA/RNA nonspecific bba-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.
Methods: To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies.
Results: Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified.
Conclusions: Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. - 中文關鍵字: --
- 英文關鍵字: --