The aim of the work presented here was the understanding of catalysis of the vitamin B1-dependent enzyme transketolase A from Escherichia coli on a molecular level. In this concern methods of molecular biology (site directed mutagenesis) were used to generate active-site variants as well as for the introduction of a sequence for a C-terminal His6-tag. By comparison of steady-state (activity, analysis of covalent intermediates) as well as pre-steady-state investigations (stopped-flow absorbance, analysis of covalent intermediates) the influence of active-site residues on catalysis were examined. The application of X-ray crystallography with substrate-soaking technique allowed the crystallization of the enzyme-bound covalent donor intermediates of D-xylulose 5-phosphate and D-fructose 6-phosphate as well as the non-covalent acceptor-substrate-enzyme-complex of ribose 5-phosphate in the docking site of transketolase A. Coenzyme analogues were used to analyse different positions of bands observed in the CD spectra. Furthermore, a hitherto undescribed acetohydroxyacid synthase (AHAS)-like carboligation reaction was shown for the enzyme.