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標題: Critical Factors Governing the Difference in Antizyme-Binding Affinities between Human Ornithine Decarboxylase and Antizyme Inhibitor
作者: Liu, Yen-Chin
Liu, Yi-Liang
Su, Jia-Yang
Liu, Guang-Yaw
Hung, Hui-Chih
關鍵字: methotrexate-induced apoptosis;analytical ultracentrifugation;polyamine metabolism;angstrom resolution;homologous protein;degradation;overexpression;growth;odc;fibroblasts
Project: Plos One, Volume 6, Issue 4, Page(s) e19253.
Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 mu M, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 mu M, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 mu M, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0019253
Appears in Collections:基因體暨生物資訊學研究所

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