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Biochemistry (Moscow)

, Volume 74, Issue 3, pp 293–300 | Cite as

New function of the amino group of thiamine diphosphate in thiamine catalysis

  • L. E. MeshalkinaEmail author
  • G. A. Kochetov
  • G. Hubner
  • K. Tittmann
  • R. Golbik
Article

Abstract

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4′-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase—4′-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4′-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4′-methylamino-ThDP-synthase activity. Thus, the 4′-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

Key words

transketolase thiamine diphosphate dihydroxyethyl-thiamine diphosphate 4′-methylamino-thiamine diphosphate stopped-flow spectroscopy CD NMR 

Abbreviations

CD

circular dichroism

DHEThDP

dihydroxyethyl-thiamine diphosphate

GA

glycolaldehyde

GAPD

glyceraldehyde-3-phosphate dehydrogenase

HP

hydroxypyruvate

ThDP

thiamine diphosphate

TK

transketolase

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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • L. E. Meshalkina
    • 1
    Email author
  • G. A. Kochetov
    • 1
  • G. Hubner
    • 2
  • K. Tittmann
    • 2
  • R. Golbik
    • 2
  1. 1.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Institute of Biochemistry/BiotechnologyMartin-Luther-University Halle-WittenbergHalle/SaaleGermany

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