| Summary: | 博士 === 國立中興大學 === 生命科學系所 === 98 === 1.Malic enzyme (ME) is a oxidative decarboxylase that catalysis a reversible reaction of malate to pyruvate, accompanying a reduction of NAD(P)+ to NADPH. There are three isoforms in mammalian cells, both mitochondria and cytosolic NADP+-dependent MEs (m-NADP-ME and c-NADP-ME) utilize NADP+ as the cofactor, and m-NAD(P)-ME is unique isoform that displayed a dual cofactor specificity, but prefers NAD+ under physiological condition. In this studies, we demonstrated the decisive amino acids (346, 347 and 362) in human m-NAD(P)-ME to govern the cofactor specificity, and successfully shifted the cofactor preference to be a NADP+-dependent enzyme. Most considerable residue for cofactor shifting was 362, and all mutants containing Q362K possessed a greater kcat,NADP value in comparison of the kcat,NAD value. The mutation of K346S and Y347K played the assistant role to promote the capacity of cofactor-shifting, and the influence of Y347K was more important than that of K346S. Additionally, the mutant K346S obviously decreased the inhibition of ATP, with a large Ki,ATP value, suggesting that residue 346 in human m-NAD(P)-ME is significant for the isoform-specific ATP inhibition. Non-cooperativity of malate binding and non-allosteric fumarate activation were other characters for m-NAD(P)-ME mutants. Overall, we provided the kinetic evidences to examine the regulative factors for cofactor specificity, and K346S/Y347K/Q362K m-NAD(P)-ME was the powerful mutant in our studies.
2.Polyamines (putrescine, spermidine, and spermine) are small, positively charged molecules that permit cells to regulate DNA, RNA, and protein involved in the cell cycle, apoptosis, protein synthesis, cell proliferation and neoplastic transformation. Ornithine decarboxylase (ODC), a pyridoxal phosphate (PLP)-dependent enzyme, catalyzes the transformation of ornithine to putrescine and is a rate-limiting step for polyamine homeostasis. Human ODC is a short-lived protein controlled by antizyme (AZ) with ubiquitin-independent degradation. AZ can interact with ODC to form an AZ/ODC heterodimer, diminishing ODC homodimer formation and inhibiting ODC enzyme activity. Antizyme inhibitor (AZI) is a homologous protein of ODC without any decarboxylating activity, and it has higher affinity than ODC to interact with AZ and to restrict AZ/ODC heterodimer formation. Therefore, ODC can recover its enzyme activity and supply putrescine again, effectively regulating polyamine. In this study, we have focused on the interaction between ODC and AZ with different lengths of AZ segments to delineate the important regions and dominant amino acids in human AZ binding site, and to confirm that AZI is behaving in the same fashion with the major binding region of AZ. We can also elucidate whether the AZ binding region commands the ODC quaternary structure or not. Finally, we will use the AZ binding region to design a functional peptide drug for antineuoplatic therapy in connection with ODC and polyamines.
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