Examination of Donor Substrate Conversion in Yeast Transketolase

Abstract The cleavage of the donor substrated-xylulose 5-phosphate by wild-type and H263A mutant yeast transketolase was studied using enzyme kinetics and circular dichroism spectroscopy. The enzymes are able to catalyze the cleavage of donor substrates, the first half-reaction, even in the absence of any acceptor substrate yielding d-glyceraldehyde 3-phosphate as measured in the coupled optical test according to Kochetov (Kochetov, G. A. (1982) Methods Enzymol. 90, 209–223) and compared with the H263A variant. Overall, the H263A mutant enzyme is less active than the wild-type. However, an increase in the rate constant of the release of the enzyme-bound glycolyl moiety was observed and related to a stabilization of the “active glycolaldehyde” (α-carbanion) by histidine 263. Chemically synthesized dl-(α,β-dihydroxyethyl)thiamin diphosphate is bound to wild-type transketolase with an apparentK D of 4.3 ± 0.8 μm (racemate) calculated from titration experiments using circular dichroism spectroscopy. Both enantiomers are cleaved by the enzyme at different rates. In contrast to the enzyme-generated α-carbanion of (α,β-dihydroxyethyl)thiamin diphosphate formed by decarboxylation of hydroxylactylthiamin diphosphate after incubation of transketolase with β-hydroxypyruvate, the synthesizeddl-(α,β-dihydroxyethyl)thiamin diphosphate did not work as donor substrate when erythrose 4-phosphate is used as acceptor substrate in the coupled enzymatic test according to Sprenger (Sprenger, G. A., Schorken, U., Sprenger, G., and Sahm, H. (1995) Eur. J. Biochem. 230, 525–532).