An efficient amperometric transketolase assay: towards inhibitor screening

Area of Science:

• Biochemistry
• Enzyme kinetics
• Biosensor technology

Background:

• Transketolase (TK) is crucial in the pentose phosphate pathway.
• Accurate determination of TK activity is vital for understanding metabolic processes.
• Existing methods for TK activity assay can be limited in sensitivity or reusability.

Purpose of the Study:

• To develop an innovative amperometric biosensor for quantifying transketolase activity from Escherichia coli (TKec).
• To establish a sensitive and reusable bienzymatic sensing system for TKec.
• To validate the biosensor's performance by determining cofactor binding and substrate/inhibitor interactions.

Main Methods:

• Immobilization of galactose oxidase (GAOx) in laponite clay for L-erythrulose detection.
• Development of a bienzymatic system with immobilized apoenzyme TKec and GAOx.
• Amperometric detection of TKec activity using d-fructose-6-phosphate and glycolaldehyde.
• Validation using thiamine diphosphate (cofactor) and substrate/inhibitor analogs.

Main Results:

• A calibration curve for TKec concentration (0.01–0.1 U ml⁻¹) was established.
• The bienzymatic biosensor demonstrated enhanced sensitivity and reusability.
• The system successfully determined the apparent dissociation constant (KD(app)) for thiamine diphosphate.
• Inhibition studies with pyrophosphate and d-arabinose-5-phosphate were performed.

Conclusions:

• The developed amperometric biosensor provides a reliable method for in vitro determination of TKec activity.
• The bienzymatic approach enhances sensitivity and reusability, offering advantages over traditional methods.
• This biosensor platform is suitable for studying TK cofactor interactions and inhibitor effects.