title: Determinants of the differential antizyme-binding affinity of ornithine decarboxylase.
abstract: Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K(d) value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K(d) value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K(d) was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K(d), which suggests that residues 119 and 137 play a role in AZ binding.
authors.affiliation: Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan. liugy@csmu.edu.tw
authors.forename: Yen-Chin
authors.initials: YC
authors.lastname: Liu
authors.forename: Den-Hua
authors.initials: DH
authors.lastname: Hsu
authors.forename: Chi-Liang
authors.initials: CL
authors.lastname: Huang
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Liu
authors.forename: Guang-Yaw
authors.initials: GY
authors.lastname: Liu
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D000818:Animals; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D014461:Ultracentrifugation
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; C031017:ornithine decarboxylase antizyme
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