Characterization of the role of Lys 69 and Cys 360 in the free radical tolerance and protein stability of human ornithine decarboxylase

• Main Authors: Yi-Chin Yeh, 葉奕辰
• Other Authors: Hui-Chih Hung
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/58498791883641704408

碩士 === 國立中興大學 === 生命科學系 === 93 === The human Ornithine decarboxylase (EC 4. 1. 1. 17; ODC) catalyzes the decarboxyation of L-ornithine to form putrescine. The monomer of the enzyme has 461 amino acids. Human ODC is an obligate homodimer with the two identical active sites formed at the interface between the monomers. The PLP cofactor is bound in a Schiff base linkage to Lys69 and it is likely that Cys360 plays an essential role in ensuring correct protonation of the decarboxylated reaction intermediate at Cα. The products usually referred to as physiological polyamines are putrescine (1.4-diminobutane), spermidine and spermine. Polyamines play many roles in cellular physiology including the ability to selectively alter transcription and translation of many genes, modulation of enzyme activities, regulation of ion channels and the formation of hypusine. In this study, we are interested in studying the effect of free radical tolerance and stability on mutations at active site residues ODC. Frist, we created three ODC mutants (K69A, C360A, K69A/C360A). Our experiments contain: Cu2+ tests and vitamin C doses tests, ornithine, DFMO, PLP, putrescine, PLP analogous doses test, protection tests. We describe the free radical tolerance and stability of wild type and mutantions by using SDS-PAGE analysis. Our data indicated that PLP has the best antioxidative effect compares with ornithine, DFMO, putrescine, and the PLP analogs. Moreover, compared with DFMO, ornithine and the putrescine dose experiment, we found that these three compounds protect effect of ODC is different. Degradation studies clearly showed the differences in free radical tolerance and stability among the ODC wild type and three mutants, K69A, C360A, K69A/C360A. The order is (wild type ≒ C360A ＞ K69A/C360A ＞ K69A).