COMPARISON OF THE BINDING OF BETA-CYCLODEXTRIN AND ALPHA-CYCLODEXTRIN AND GAMMA-CYCLODEXTRIN 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, K-d, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, K-i, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The K-d 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. Preliminary kinetic studies were done on the binding of the enzyme with alpha-, beta-, and gamma-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, k(app) for alpha- and gamma-cyclodextrins were independent of cyclodextrin concentration in the range studied (0.05-1.25 mM). In contrast, beta-cyclodextrin showed a hyperbolic concentration dependence of k(app) 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.