Abstract
Glucoamylase I (GAI) bound to β-cyclodextrin (β-CD) with the binding constants of Kd = 17.7μM and n = 1.69. Binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for β-CD inhibited the binding of GAI to β-CD. Titration of GAI (A1-R615) with β-CD or Amylose A showed critical spectral perturbations at 286 nm that reflected the aromatic side chains of Tyr and Trp, but no spectral changes were observed in GAI' (A1-V469) and Gp-I (A470-V514). Titration of GAI with derivatives of β-CD, namely, 6-hydroxypropyl-β-CD (H-CD), 2, 6-O-dimethyl-β-CD, and 2, 3, 6-O-trimethyl-β-CD, yielded spectral changes only in the case of H-CD. Therefore, the Cp region (A515-R615) of GAI seems stereospecifically able to recognize the structure of the secondary OH-side but not the primary OH-side of β-CD. Chemical modifications specific for aromatic amino acids of the GAI-CD inclusion complexes were done with N-bromosuccinimide and tetranitromethane. In the presence of β-CD, one Trp residue was protected from oxidation but Tyr residues were not. The analysis of a Cp region/β-galactosidase fusion protein indicated that Trp562 contributed to formation of inclusion complexes.
