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    National Tsing Hua University Institutional Repository > 生命科學院  > 生命科學系 > 期刊論文 >  Activation of Helicobacter pylori inorganic pyrophosphatase and the importance of Cys16 in thermostability, enzyme activation and quaternary structure

    Please use this identifier to cite or link to this item: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/49180

    Title: Activation of Helicobacter pylori inorganic pyrophosphatase and the importance of Cys16 in thermostability, enzyme activation and quaternary structure
    Authors: Lee, Mon Juan;Huang, Haimei;Lin, Wei;Yang, Ray Rong;Liu, Chien Liang;Huang, Chung Yu
    教師: 黃海美
    Date: 2007
    Publisher: Springer Verlag
    Relation: ARCHIVES OF MICROBIOLOGY,Springer Verlag,Volume 188,Issue 5,NOV 2007,Pages 473-482
    Keywords: reductants
    site-directed mutagenesis
    Helicobacter pylori
    inorganic pyrophosphatase (PPase)
    Abstract: The inorganic pyrophosphatase from the human pathogen Helicobacter pylori (HpPPase) is a family I PPase. It is a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of inorganic pyrophosphate (PPi) by HpPPase relied on the presence of magnesium and followed Michaelis-Menten kinetics, with k (cat) being 344 s(-1) and K-m being 83 mu M at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substitution of Cys16 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C16S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y140F HpPPase and those of Escherichia coli. Although Cys16 was not located at the subunit interface of the hexameric HpPPase, sedimentation analysis results suggested that the C16S substitution destabilized HpPPase through impairing trimer-trimer interactions. This study provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure.
    URI: http://www.springerlink.com/
    Appears in Collections:[生命科學系] 期刊論文

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