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Isolation and properties of human transketolase

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Abstract

Recombinant human (His)6-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).

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Abbreviations

DHEThDP:

α,β-dihydroxyethylthiamine diphosphate

DTT:

dithiothreitol

GA:

glycolaldehyde

3-GAP:

glyceraldehyde 3-phosphate

GAPD:

glyceraldehyde-3-phosphate dehydrogenase

hTK:

transketolase from normal human tissues

R-5-P:

ribose 5-phosphate

ThDP:

thiamine diphosphate

TK:

transketolase

TKTL1:

transketolase from human tumor tissues

X-5-P:

xylulose 5-phosphate

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Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    L. E. Meshalkina, O. N. Solovjeva, Yu. A. Khodak, V. L. Drutsa & G. A. Kochetov

Authors
  1. L. E. Meshalkina
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  2. O. N. Solovjeva
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  3. Yu. A. Khodak
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  4. V. L. Drutsa
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  5. G. A. Kochetov
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Correspondence to L. E. Meshalkina.

Additional information

Published in Russian in Biokhimiya, 2010, Vol. 75, No. 7, pp. 992–1000.

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Meshalkina, L.E., Solovjeva, O.N., Khodak, Y.A. et al. Isolation and properties of human transketolase. Biochemistry Moscow 75, 873–880 (2010). https://doi.org/10.1134/S0006297910070096

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  • DOI: https://doi.org/10.1134/S0006297910070096

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