Researcher:
Koper, Kaan

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Master Student

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Kaan

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Koper

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Koper, Kaan

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2014 - 20162

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Researcher Of Publications: search.filters.isAuthorOfPublication.d378567d-2f20-41ad-8577-a361baa7858a

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    Glu-370 in the large subunit influences the substrate binding, allosteric, and heat stability properties of potato ADP-glucose pyrophosphorylase
    (Elsevier Ireland Ltd, 2016) Çalışkan, Mahmut; Cevahir, Gül; N/A; Department of Chemical and Biological Engineering; N/A; Department of Molecular Biology and Genetics; N/A; Department of Chemical and Biological Engineering; Seferoğlu, Ayşe Bengisu; Gül, Şeref; Dikbaş, Uğur Meriç; Barış, İbrahim; Koper, Kaan; Kavaklı, İbrahim Halil; PhD Student; Researcher; Master Student; Teaching Faculty; Master Student; Faculty Member; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Graduate School of Sciences and Engineering; College of Engineering; Graduate School of Sciences and Engineering; College of Sciences; Graduate School of Sciences and Engineering; College of Engineering; N/A; 289253; N/A; 111629; N/A; 40319
    ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the (LSSSWT)-S-E370G AGPase for glucose 1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the (LSSSWT)-S-E370G AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LS(E370G)SS(WT)AGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.
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    Enhanced heterotetrameric assembly of potato ADP-Glucose pyrophosphorylase using reverse genetics
    (Oxford Univ Press, 2014) Cevahir, Gül; N/A; N/A; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Seferoğlu, Ayşe Bengisu; Koper, Kaan; Can, Fatma Betül; Kavaklı, İbrahim Halil; PhD Student; Master Student; Undergraduate Student; Faculty Member; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; College of Sciences; College of Engineering; N/A; N/A; N/A; 40319
    ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. Computational and experimental studies have revealed that the heterotetrameric assembly of AGPase is thermodynamically weak. Modeling studies followed by the mutagenesis of the LS of the potato AGPase identified a heterotetramer-deficient mutant, LSR88A. To enhance heterotetrameric assembly, LSR88A cDNA was subjected to error-prone PCR, and second-site revertants were identified according to their ability to restore glycogen accumulation, as assessed with iodine staining. Selected mutations were introduced into the wild-type (WT) LS and co-expressed with the WT SS in Escherichia coli glgC(-). The biochemical characterization of revertants revealed that (LSSSWT)-S-I90V, (LSSSWT)-S-Y378C and (LSSSWT)-S-D410G mutants displayed enhanced heterotetrameric assembly with the WT SS. Among these mutants, (LSSSWT)-S-Y378C AGPase displayed increased heat stability compared with the WT enzyme. Kinetic characterization of the mutants indicated that the (LSSSWT)-S-I90V and (LSSSWT)-S-Y378C AGPases have comparable allosteric and kinetic properties. However, the (LSSSWT)-S-D410G mutant exhibited altered allosteric properties of being less responsive and more sensitive to 3-phosphoglyceric acid activation and inorganic phosphate inhibition. This study not only enhances our understanding of the interaction between the SS and the LS of AGPase but also enables protein engineering to obtain enhanced assembled heat-stable variants of AGPase, which can be used for the improvement of plant yields.