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J. Biol. Chem., Vol. 276, Issue 19, 16051-16058, May 11, 2001
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From the The cleavage of the donor substrate
D-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" (
Examination of Donor Substrate Conversion in Yeast
Transketolase*
§,,§,,, and
Institute of Biochemistry, Department of
Biochemistry and Biotechnology, Martin-Luther-University
Halle-Wittenberg, 06120 Halle/Saale, Kurt-Mothes-Stra
e 3, Germany
and the ¶ Division of Molecular Structural Biology, Department of
Medical Biochemistry and Biophysics, Karolinska Institute,
Tomtebodavägen 6, 171 77 Stockholm, Sweden
-carbanion) by histidine 263. Chemically synthesized DL-(,-dihydroxyethyl)thiamin diphosphate
is bound to wild-type transketolase with an apparent
KD 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 -carbanion of
(,-dihydroxyethyl)thiamin diphosphate formed by decarboxylation
of hydroxylactylthiamin diphosphate after incubation of transketolase
with -hydroxypyruvate, the synthesized
DL-(,-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., Schörken, U., Sprenger, G., and Sahm, H. (1995) Eur. J. Biochem. 230, 525-532).
*
This work was supported by the Deutsche
Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the
Swedish Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
49-345-5524828; Fax: 49-345-5527011; E-mail:
huebner@biochemtech. uni-halle.de.
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