| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 97 === Human ornithine decarboxylase (hODC) is a pyrodoxal 5’-phosphate (PLP)-dependent enzyme that involves in polyamine biosynthesis and catalyzes polyamine formation. The catalytically active form of hODC is dimeric form. AZ has been identified its role in facilitating degradation of mammalian ODC. Binding of antizyme promotes the dissociation of ODC homodimers and targets ODC for degradation by the 26S proteasomes. In contrast, trypanosomal ODC (tODC) cannot bind to AZ. In this study, we aim to identify the essential amino acid residues governing the AZ-binding affinity for ODC. Based on the multiple sequence alignments in the putative AZ-binding site of ODC, the non-conserved amino acid residues on tODC will be introduced into hODC. If the mutant decreased its AZ-binding affinity, the ODC enzyme activity will not be largely reduced in the presence of AZ. Our data indicated that the single mutant, Q119H, Q129D, V137D and M140E demonstrated a higher residual enzyme activity, suggesting that the additional charge of the three residues will disadvantage the AZ binding. Furthermore, the multiple mutants displayed insensitivity toward AZ inhibition. According to our results, we can identify the essential amino acid residues on hODC required for AZ binding. We can assess the Kd value between ODC and AZ by analytical ultracentrifugation and the results are consistent.
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