Dissociation/association properties of a dodecameric cyclomaltodextrinase: Effects of pH and salt concentration on the oligomeric state

Hee Seob Lee, Jin Soo Kim, Kyuho Shim, Jung Woo Kim, Kuniyo Inouye, Hiroshi Oneda, Young Wan Kim, Kyung Ah Cheong, Hyunju Cha, Eui Jeon Woo, Joong Hyuck Auh, Sung Joon Lee, Jung Wan Kim, Kwan Hwa Park

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14 Citations (Scopus)

Abstract

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I-5 (CDase I-5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I-5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain-swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I-5 was present as a dimer in the pH range of 5.0-6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I-5 dissociated into dimers at a rate of 8.58 × 10-2 h-1 at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild-type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH-dependent process, the dissociation of wild-type CDase I-5 proceeded very fast at pH 7.0 in the presence of 0.2-1.0 m of KCl. Stopped-flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s-1 and 7.99 s-1 in the presence of 0.2 m and 1.0 m of KCl, respectively.

Original languageEnglish
Pages (from-to)109-121
Number of pages13
JournalFEBS Journal
Volume273
Issue number1
DOIs
Publication statusPublished - 2006 Jan

Keywords

  • Cyclomaltodextrinase
  • Dissociation/ association
  • Dodecamer
  • Maltogenic amylase
  • Oligomerization
  • Quaternary structure

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Lee, H. S., Kim, J. S., Shim, K., Kim, J. W., Inouye, K., Oneda, H., Kim, Y. W., Cheong, K. A., Cha, H., Woo, E. J., Auh, J. H., Lee, S. J., Kim, J. W., & Park, K. H. (2006). Dissociation/association properties of a dodecameric cyclomaltodextrinase: Effects of pH and salt concentration on the oligomeric state. FEBS Journal, 273(1), 109-121. https://doi.org/10.1111/j.1742-4658.2005.05047.x