THE MECHANISM OF BINDING OF GLUCOAMYLASE-I FROM ASPERGILUS-AWAMORI VAR KAWACHI TO CYCLODEXTRINS AND RAW STARCH

Glucoamylase I (GAI) bound to beta-cyclodextrin (beta-CD) with the binding constants of K-d = 17.7 mu M and n = 1.69. Binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for beta-CD inhibited the binding of GAI to beta-CD. Titration of GAI (A(1)-R(615)) with beta-CD or Amylose A shelved critical spectral perturbations at 286 nm that reflected the aromatic side chains of Tyr and Trp, but no spectral changes were observed in GAI (A(1)-V-469) and Gp-I (A(470)-V-514). Titration of GAI with derivatives of beta-CD, namely, 6-hydroxpropyl-beta-CD (H-CD), 2,6-O-dimethyl-beta-CD, and 2,3,6-O-trimethyl-beta-CD, yielded spectral changes only in the case of H-CD. Therefore, the Cp region (A(515)-R(615)) of GAI seems stereospecifically able to recognize the structure of the secondary OH-side but not the primary OH-side of beta-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 beta-CD, one Trp residue was protected from oxidation but Tyr residues were not. The analysis of a Cp region/beta-galactosidase fusion protein indicated that Trp(562) contributed to formation of inclusion complexes.