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Science 3 January 1997:
Vol. 275. no. 5296, pp. 67 - 70
DOI: 10.1126/science.275.5296.67
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Reports

How Thiamine Diphosphate Is Activated in Enzymes

Dorothee Kern, *dagger Gunther Kern, ddagger Holger Neef, Kai Tittmann, Margrit Killenberg-Jabs, Christer Wikner, Gunter Schneider, Gerhard Hübner

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

D. Kern, G. Kern, H. Neef, K. Tittmann, M. Killenberg-Jabs, G. Hübner, Institut für Biochemie, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle, Germany.
C. Wikner and G. Schneider, Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Doktorsringen 4, S-17177 Stockholm, Sweden.
*   To whom correspondence should be addressed.

dagger    Present address: Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA.

ddagger    Present address: Department of Molecular and Cellular Biology, University of California at Berkeley, Berkeley, CA 94720, USA.

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From the Cover: Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the alpha -carbanion of (alpha ,beta -dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae.
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