Examination of donor substrate conversion in yeast transketolase.
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" (alpha-carbanion) by histidine 263. Chemically synthesized dl-(alpha,beta-dihydroxyethyl)thiamin diphosphate is bound to wild-type transketolase with an apparent K(D) of 4.3 +/- 0.8 microm (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 alpha-carbanion of (alpha,beta-dihydroxyethyl)thiamin diphosphate formed by decarboxylation of hydroxylactylthiamin diphosphate after incubation of transketolase with beta-hydroxypyruvate, the synthesized dl-(alpha,beta-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).
Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle/Saale, Kurt-Mothes-Strabetae 3, Germany.
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E
Fiedler
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Golbik
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Schneider
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Tittmann
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Neef
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König
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Hübner
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D005636:Fructosephosphates / Q000378:metabolism; D014644:Genetic Variation; D006639:Histidine; D007700:Kinetics; D015394:Molecular Structure; D010428:Pentosephosphates / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014174:Transketolase / Q000737:chemistry* / Q000378:metabolism*
D005636:Fructosephosphates; D010428:Pentosephosphates; D011994:Recombinant Proteins; C093420:dihydroxyethylthiamine pyrophosphate; D006639:Histidine; C031625:xylulose-5-phosphate; C027618:fructose-6-phosphate; D014174:Transketolase; D013835:Thiamine Pyrophosphate