Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy

Mariya Kokova, Michael Zavrel, Kai Tittmann, Antje C. Spiess, Martina Pohl

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

The benzoin condensation reaction catalyzed by the thiamine diphosphate (ThDP)-dependent enzymes benzaldehyde lyase (BAL) and benzoylformate decarboxylase variant His281Ala (BFDH281A) was studied via initial rate measurements, progress curve analysis and NMR-based analysis of reaction intermediates. Using a mechanistic kinetic model, the kinetic parameters and microscopic rate constants were determined, thus identifying the rate limiting steps of the reaction. In BAL, overall reaction is rate-limited by product release, whereas in BFDH281A substrate binding is the slowest step of catalysis. These results were further confirmed by analysis of covalent reaction intermediates using NMR spectroscopy after acid quench isolation.

Original languageEnglish
Pages (from-to)73-79
Number of pages7
JournalJournal of Molecular Catalysis B: Enzymatic
Volume61
Issue number1-2
DOIs
StatePublished - Nov 2009
Externally publishedYes

Keywords

  • Carboligation
  • Intermediates
  • Kinetic modeling
  • NMR spectroscopy
  • Reaction mechanism
  • Thiamine diphosphate-dependent enzymes

Fingerprint

Dive into the research topics of 'Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy'. Together they form a unique fingerprint.

Cite this