NMR analysis of covalent intermediates in thiamin diphosphate enzymes

Kai Tittmann, Ralph Golbik, Kathrin Uhlemann, Ludmila Khailova, Gunter Schneider, Mulchand Patel, Frank Jordan, David M. Chipman, Ronald G. Duggleby, Gerhard Hübner

Research output: Contribution to journalArticlepeer-review

130 Scopus citations

Abstract

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using 1H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Original languageEnglish
Pages (from-to)7885-7891
Number of pages7
JournalBiochemistry
Volume42
Issue number26
DOIs
StatePublished - 8 Jul 2003

ASJC Scopus subject areas

  • Biochemistry

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