Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192: nitration with tetranitromethane

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans is crucial for carbohydrate processing.
• Understanding enzyme structure-function relationships, particularly the role of tyrosine residues, is vital for enzyme engineering.

Purpose of the Study:

• To investigate the role of tyrosine residues in CGTase activity and structure.
• To determine the effect of nitration on CGTase conformation and function.
• To explore the potential involvement of tyrosine in the catalytic site.

Main Methods:

• Chemical modification of Bacillus circulans CGTase using tetranitromethane (TNM).
• Spectroscopic analysis (fluorescence) to monitor enzyme conformation.
• Enzyme kinetics and activity assays.
• Investigation of acarbose's protective effect during nitration.
• Analysis of enzyme affinity for beta-cyclodextrin co-polymer.

Main Results:

• Nitration modified up to 15 of 28 tyrosine residues, leading to loss of enzymatic activity and tryptophan fluorescence.
• Impairment of enzyme conformation was observed due to tyrosine nitration.
• Acarbose protected one tyrosine residue, reducing the inactivation rate 9.4-fold, indicating a catalytic site tyrosine.
• CGTase affinity for beta-cyclodextrin decreased, attributed to a side reaction at the raw-starch-binding site, not tyrosine modification.

Conclusions:

• Tyrosine residues play a significant role in maintaining CGTase conformation and activity.
• Evidence supports the involvement of at least one tyrosine residue in the CGTase catalytic mechanism.
• The observed decrease in beta-cyclodextrin binding is likely due to a separate modification at the starch-binding site.