| Summary: | 博士 === 國立中興大學 === 生命科學系所 === 97 === English Abstract
Human ornithine decarboxylase (ODC; EC 4.1.1.17) is a pyrodoxal 5’-phosphate (PLP) dependent enzyme that catalyzes the first and rate-limiting step in polyamine biosynthesis. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface between the monomers. Antizyme (AZ) interacts with ODC to form heterodimer that is then degraded by 26S proteasome without ubiquitin binding. Human antizyme inhibitor (AZI) is highly homologous to ODC but lacks enzymatic activity, and it exists as a monomer. AZI has higher affinity toward AZ than ODC and that may rescue ODC enzyme activity. In this study, we aim to identify the essential amino acid residues governing the quaternary structure formation for ODC and AZI. Based on the multiple sequence alignments of ODC and AZI, the non-conserved amino acid residues in the putative dimer-interface of AZI (S277, S331, E332 and D389) were changed to the amino acid residues of ODC (R277, Y331, D332 and Y389, respectively). Analytical ultracentrifugation were used to obtain the size-distribution of these AZI mutants. Sedimentation velocity and continuous size distribution analysis demonstrated that the wild-type ODC (ODC-wt) is a dimer with a disassociation constant (Kd) of 0.18 μM. In contrast, the wild-type AZI (AZI-wt) displayed as a monomer with a Kd of 84.0 μM. The AZI-S331Y mutant displayed a monomer-dimer equilibrium with a Kd of 40.7 μM showing a lower Kd than that of AZI-wt. The double mutant, AZI-S331Y-D389Y, triple mutant, AZI-S331Y-D389Y-S277R and quadruple mutant, AZI-S331Y-D389Y-S277R-E332D displayed as a dimer with a Kd value of 2.7 μM, 1.3 μM and 0.10 μM, respectively, close to that of ODC-wt. The AZI-mutants without S331Y showed similar Kd values to that of AZI-wt. Our findings indicated that the residue 331 play a key role determining the dimer formation of ODC and AZI.
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