Isolation and properties of human transketolase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Transketolase (TK) is a crucial enzyme in the pentose phosphate pathway.
• Understanding human transketolase (hTK) function requires pure enzyme preparations.
• Thiamine diphosphate (ThDP) is an essential cofactor for transketolase activity.

Purpose of the Study:

• To produce recombinant human transketolase (hTK) in large quantities.
• To develop methods for isolating both holoenzyme and apoenzyme forms of hTK.
• To characterize the kinetic properties of hTK, including substrate and cofactor interactions.

Main Methods:

• Heterologous expression of the human transketolase gene in Escherichia coli.
• Purification of recombinant hTK using affinity chromatography.
• SDS-PAGE for purity assessment, circular dichroism (CD) spectroscopy for cofactor binding and reaction monitoring, and kinetic assays.

Main Results:

• Homogeneous recombinant hTK (holoenzyme) was obtained in preparative amounts.
• A method for generating the hTK apoenzyme was established.
• CD spectroscopy confirmed ThDP binding to the apoenzyme and allowed real-time reaction monitoring; Michaelis constants for ThDP and physiological substrates were determined.

Conclusions:

• Recombinant expression provides a viable source of pure hTK.
• The developed methods enable detailed biochemical and kinetic studies of hTK.
• Characterization of hTK kinetics provides insights into its role in metabolic pathways.