INVESTIGATION OF THE ACTIVE-SITE OF BACILLUS-MACERANS CYCLODEXTRIN GLUCANOTRANSFERASE BY USE OF MODIFIED MALTOOLIGOSACCHARIDES

The active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl-alpha-maltopentaoside and phenyl-alpha-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1' S2', etc.) geometrically complementary to several glucose residues, and the alpha-1,4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl alpha-maltopentaoside (G-G-G-G-G-PHI; G, glucose residue; PHI, phenyl residue; -, alpha-1,4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-PHI; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH, (AG-G-G-G-G-PHI; AG, 6-amino-6-deoxy-D-glucose residue), or COOH (CG-G-G-G-G-PHI; CG, glucuronic acid residue). p-Nitrophenyl alpha-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-PHI was excluded from subsite S3, while it was accommodated in subsite S4. The K(m) and V(max) values for CG-G-G-G-G-PHI were remarkably larger and smaller, respectively, than those for any other substrates. The results suggested that the CH2R groups (R:OH,I,NH2) have interactions with subsite S3 and/or the COOH group interrupts binding of the CG residue to subsite S3. The features of subsite S1' were examined using phenyl-alpha-D-glucopyranoside derivatives [G-PHI, IG-PHI, DG-PHI (DG: 6-deoxy-D-glucose residue), AG-PHI, or CG-PHI] as acceptors. FG4P (FG-G-G-G-P, FG: 6-deoxy-6- [ (2-pyridyl)amino ] -D-glucose residue) was used as the substrate. The product analysis showed that the extent of transfer reaction to produce FG-G-G-XG-PHI decreased in the order of G-PHI, IG-PHI, DG-PHI, AG-PHI, and CG-PHI, and the extent of hydrolysis to give FG-G-G and GP increased in the same order. These data suggested that subsite S1' excluded the charged groups, especially negatively charged groups, of modified acceptors, and CGTase catalyzed mainly the transfer reaction in the presence of a suitable acceptor, and the hydrolysis reaction with unsuitable acceptors.