The active site of cyclodextrin glycosyltransferase from Bacillus sp. A11

Abstract

Chemical modification of CGTase from Bacillus sp. A11 was performed under optimized mild conditions. Loss of CGTase activities was observed when modifications were made on carboxyl, histidine, tryptophan, and tyrosine residues by treatment with 1 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), diethylpyrocarbonate (DEP), N-bromosuccinimide (NBS) an dN-acetylimidazole (NAI), respectively. While modification of cysteine, lysine, and serine residues with up to 100 mM concentrations of N-ethylmaleimide (NEM), 2, 4, 6-trinitrobenzenesulfonic acid, and phenylmethylsulfonyl fluoride, respectively did not affect CGTase activities. For cysteine modification, iodoacetamide and dithiothreitol treatment gave the same result as NEM. Protection of the active site of CGTase with protective substance (alpha-, beta-, gamma-CD, or maltotriose) prior to chemical modification significantly reduced activity loss. These results suggested that carboxyl (aspartic and glutamic acids), histidine, tryptophan, and tyrosine residues were located at the active site of CGTase. In the estimation of the number of histidine, tryptophan, and tyrosine residues at the active site of the enzyme using spectrophotometric determination, it was found that beta-CD or gamma-CD protects two histidine residues, one tryptophan residue, and two tyrosine residues of CGTase. This suggests the presence of these amino acid residues at the active site of CGTase. By non-denaturing polyacrylamide gel electrophoresis, effect of chemical modification on the enzyme structure was analyzed. The modification of carboxyl groups by EDC resulted in a protein band which moved slower than the unmodified enzyme suggesting that the net negative charges of the enzyme were reduced. Modification of tryptophan by NBS did not change the pattern of protein and activity bands in the gel. When the enzyme was modified by DEP or NAI, faster protein and activity bands were observed. The result suggests that these two reagents induced more net negative charges or structural changes which leads to more exposed negative charges. Analysis by PAGE thus confirms the modification on carboxyl, histidine, tryptophan, and tyrosine residues by EDC, DEP, NBS, and NAI, respectively. When kinetic parameters of CGTase for cyclodextrin substrates were measured, the michaelis constant (Km) values for coupling reaction were 1.55, 1.60, and 1.94 mM and Vmax values were 2.81, 2.50, and 1.40 mumoles/min for beta-CD, maltosyl (G2)-beta-CD, and methyl-beta-CD, respectively. For cyclodextrin degrading activity, the Km values were 3.16, 1.69, 1.42, and 91.63 mM and Vmax values were 58.96, 13.12, 8.61, and 10.69 mumoles/min for alpha-, beta-, gamma-CD, and maltotriose, respectively. The result indicates that beta- and gamma-CD were more suitable for substrate binding site than the smaller molecules, alpha-CD or linear maltotriose.