title: Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state.
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 x 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.
authors.affiliation: Center for Agricultural Biomaterials, and School of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
authors.forename: Hee-Seob
authors.initials: HS
authors.lastname: Lee
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.forename: Kyuho
authors.initials: K
authors.lastname: Shim
authors.forename: Jung-Woo
authors.initials: JW
authors.lastname: Kim
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Oneda
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.forename: Kyung-Ah
authors.initials: KA
authors.lastname: Cheong
authors.forename: Hyunju
authors.initials: H
authors.lastname: Cha
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.forename: Joong Hyuck
authors.initials: JH
authors.lastname: Auh
authors.forename: Sung-Joon
authors.initials: SJ
authors.lastname: Lee
authors.forename: Jung-Wan
authors.initials: JW
authors.lastname: Kim
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D002850:Chromatography, Gel; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000378:metabolism / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D006639:Histidine; D011189:Potassium Chloride; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase