Comparison of the binding of beta-cyclodextrin and alpha- and gamma-cyclodextrins with pullulanase from Klebsiella pneumoniae as studied by equilibrium and kinetic fluorometry.
The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was investigated at 25 degrees C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied. The dissociation constant, Kd, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, Ki, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The Kd values of beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin were approximately two orders of magnitude smaller than those of alpha- and gamma-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among alpha-, beta-, and gamma-cyclodextrins for binding with the enzyme, which indicates that the binding region of beta-cyclodextrin overlaps those of alpha- and gamma-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for alpha- and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin, whereas beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of beta-cyclodextrin is slightly different from those of alpha- and gamma-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)
Iwamoto
H
H
Department of Food Science and Technology, Faculty of Engineering, Fukuyama University, Hiroshima.
Ohno
M
M
Ohmori
M
M
Hirose
J
J
Tanaka
A
A
Sakai
S
S
Hiromi
K
K
D001426:Bacterial Proteins / Q000378:metabolism*; D003505:Cyclodextrins / Q000378:metabolism*; D005470:Fluorometry; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D003505:Cyclodextrins; D006026:Glycoside Hydrolases; C100181:pullulanase