Observation of thiamin-bound intermediates and microscopic rate constants for their interconversion on 1-deoxy- d -xylulose 5-phosphate synthase: 600-Fold rate acceleration of pyruvate decarboxylation by d -glyceraldehyde-3-phosphate.

Hetalben Patel, Natalia S. Nemeria, Leighanne A. Brammer, Caren L. Freel Meyers, Frank Jordan

Research output: Contribution to journalArticle

Abstract

The thiamin diphosphate (ThDP)-dependent enzyme 1-deoxy-d-xylulose 5-phosphate (DXP) synthase carries out the condensation of pyruvate as a 2-hydroxyethyl donor with d-glyceraldehyde-3-phosphate (d-GAP) as acceptor forming DXP. Toward understanding catalysis of this potential anti-infective drug target, we examined the pathway of the enzyme using steady state and presteady state kinetic methods. It was found that DXP synthase stabilizes the ThDP-bound predecarboxylation intermediate formed between ThDP and pyruvate (C2α-lactylThDP or LThDP) in the absence of d-GAP, while addition of d-GAP enhanced the rate of decarboxylation by at least 600-fold. We postulate that decarboxylation requires formation of a ternary complex with both LThDP and d-GAP bound, and the central enzyme-bound enamine reacts with d-GAP to form DXP. This appears to be the first study of a ThDP enzyme where the individual rate constants could be evaluated by time-resolved circular dichroism spectroscopy, and the results could have relevance to other ThDP enzymes in which decarboxylation is coupled to a ligation reaction. The acceleration of the rate of decarboxylation of enzyme-bound LThDP in the presence of d-GAP suggests a new approach to inhibitor design.

Original languageEnglish (US)
Pages (from-to)18374-18379
Number of pages6
JournalJournal of the American Chemical Society
Volume134
Issue number44
DOIs
StatePublished - Nov 7 2012

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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