Understanding the mechanism and structure of transketolase is valuable across a range of disciplines, including enzymology, synthetic biology, drug development, and biocatalysis. Beyond offering insights into enzyme catalysis and thiamin-dependent chemistry, this knowledge enables the rational design of transketolase variants with altered substrate specificity and the creation of novel biosynthetic pathways to produce unusual sugars or chiral compounds. Transketolase is also a potential target for cancer treatment, as well as for metabolic or neurodegenerative diseases. This work presents protocols for analyzing transketolase activity, its catalytic mechanism, and structure. These include methods for steady-state kinetics, cofactor binding, detection of catalytic intermediates, and rapid kinetic studies using spectroscopic and biophysical techniques. Together, these protocols furnish a comprehensive toolkit for advancing both fundamental and applied transketolase research.
Keywords: Cofactor; Enzymatic assay; Enzyme mechanism; Intermediates; Kinetics; Spectroscopy; Stopped-flow; Thiamin diphosphate.
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