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Examination of donor substrate conversion in yeast transketolase
E Fiedler1, R Golbik, G Schneider
1Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale, Kurt-Mothes-Strabetae 3, Germany.
The Journal of Biological Chemistry
|March 30, 2001
Summary
Wild-type and H263A mutant yeast transketolase were studied. The H263A mutant showed lower activity but stabilized the active glycolaldehyde intermediate, suggesting histidine 263 is crucial for transketolase function.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Transketolase is a key enzyme in the pentose phosphate pathway.
- Understanding its catalytic mechanism is vital for metabolic studies.
Purpose of the Study:
- To investigate the catalytic activity of wild-type and H263A mutant yeast transketolase.
- To elucidate the role of histidine 263 in the enzyme's mechanism.
Main Methods:
- Enzyme kinetics assays.
- Circular dichroism spectroscopy.
- Coupled optical tests for product quantification.
Main Results:
- The H263A mutant exhibited reduced overall activity compared to wild-type transketolase.
- A significant increase in the release rate of the enzyme-bound glycolyl moiety was observed for the H263A mutant.
- Histidine 263 appears to stabilize the active glycolaldehyde (alpha-carbanion) intermediate.
Conclusions:
- Histidine 263 plays a critical role in stabilizing the alpha-carbanion intermediate of transketolase.
- The H263A mutation affects the enzyme's catalytic efficiency and intermediate stability.