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Evidence for Dramatic Acceleration of a C−H Bond Ionization Rate in Thiamin Diphosphate Enzymes by the Protein Environment

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journal contribution
posted on 22.02.2005 by Sheng Zhang, Leon Zhou, Natalia Nemeria, Yan, Zhen Zhang, Yu Zou, Frank Jordan
The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2α-carbanion present on all such enzymes) was tested experimentally using the intermediate C2α-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2α-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D → H exchange from the C2α position of HEThDP-d4, as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 107, corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.