Dissociation/association properties of a dodecameric cyclomaltodextrinase. Effects of pH and salt concentration on the oligomeric state.
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.
Lee
Hee-Seob
HS
Center for Agricultural Biomaterials, and School of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Kim
Jin-Soo
JS
Shim
Kyuho
K
Kim
Jung-Woo
JW
Inouye
Kuniyo
K
Oneda
Hiroshi
H
Kim
Young-Wan
YW
Cheong
Kyung-Ah
KA
Cha
Hyunju
H
Woo
Eui-Jeon
EJ
Auh
Joong Hyuck
JH
Lee
Sung-Joon
SJ
Kim
Jung-Wan
JW
Park
Kwan-Hwa
KH
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
D006639:Histidine; D011189:Potassium Chloride; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase