Comparison of the Binding of β2-Cyclodextrin and α- and γ-Cyclodextrins with Pullulanase from Klebsiella pneumoniae as Studied by Equilibrium and Kinetic Fluorometry

• Published in: Journal of biochemistry (Tokyo) 1994-12, Vol.116 (6), p.1264-1268
• Main Authors: Iwamoto, Hiroyuki, Ohno, Masahiro, Ohmori, Masayuki, Hirose, Junzo, Tanaka, Akiyoshi, Sakai, Shuzo, Hiromi, Keitaro
• Format: Article
• Language: English
• Subjects: cyclodextrin; fluorometry; ligand binding; pullulanase; stopped-flow analysis
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a124673

The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of α-, β-, and γ-cyclodextrins and 6-O-α-glucosyl-α-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin was investigated at 25°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, id, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, Kl9 obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93–96]. The Kd values of β-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin were approximately two orders of magnitude smaller than those of α- and γ-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among α-, β-, and γ-cyclodextrins for binding with the enzyme, which indicates that the binding region of β-cyclodextrin overlaps those of α- and γ-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for α- and γ-cyclodextrins and 6-O-α-glucosyl-α-cyclodextrin, whereas β-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of β-cyclodextrin is slightly different from those of a- and γ-cyclodextrins. Preliminary kinetic studies were done on the binding of the enzyme with α-, β, and γ-cyclodextrins by following the increase in fluorescence intensity (excitation at 280 nm) with a micro-stopped-flow apparatus. A progress curve of single exponential type was observed for every cyclodextrin studied. The apparent first-order rate constants, kapp, for α- and γ-cyclodextrins were independent of cyclodextrin concentration in the range studied (0.05–1.25 mM). In contrast, β-cyclodextrin showed a hyperbolic concentration dependence of kapp, increasing asymptotically to the maximum value of about 2.9 s−1. Judged from these results, the rate-limiting step of the enzyme-cyclodextrin binding is considered to be a unimolecular process, probably a conformational isomerization.