title: Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.
abstract: 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 µM, 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 µM, 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 µM, 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.
authors.affiliation: Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Yen-Chin
authors.initials: YC
authors.lastname: Liu
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Liu
authors.forename: Jia-Yang
authors.initials: JY
authors.lastname: Su
authors.forename: Guang-Yaw
authors.initials: GY
authors.lastname: Liu
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D011485:Protein Binding; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011506:Proteins; D012997:Solvents; C031017:ornithine decarboxylase antizyme; D009955:Ornithine Decarboxylase