Difference FTIR Studies of Substrate Distribution in Triosephosphate Isomerase

Hua Deng, Jayson Vedad, Ruel Z.B. Desamero, Robert Callender

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP), via an enediol(ate) intermediate. Determination of substrate population distribution in the TIM/substrate reaction mixture at equilibrium and characterization of the substrate-enzyme interactions in the Michaelis complex are ongoing efforts toward the understanding of the TIM reaction mechanism. By using isotope-edited difference Fourier transform infrared studies with unlabeled and 13C-labeled substrates at specific carbon(s), we are able to show that in the reaction mixture at equilibrium the keto DHAP is the dominant species and the populations of aldehyde GAP and enediol(ate) are very low, consistent with the results from previous X-ray structural and 13C NMR studies. Furthermore, within the DHAP side of the Michaelis complex, there is a set of conformational substates that can be characterized by the different C2=O stretch frequencies. The C2=O frequency differences reflect the different degree of the C2=O bond polarization due to hydrogen bonding from active site residues. The C2=O bond polarization has been considered as an important component for substrate activation within the Michaelis complex. We have found that in the enzyme-substrate reaction mixture with TIM from different organisms the number of substates and their population distribution within the DHAP side of the Michaelis complex may be different. These discoveries provide a rare opportunity to probe the interconversion dynamics of these DHAP substates and form the bases for the future studies to determine if the TIM-catalyzed reaction follows a simple linear reaction pathway, as previously believed, or follows parallel reaction pathways, as suggested in another enzyme system that also shows a set of substates in the Michaelis complex.

Original languageEnglish (US)
Pages (from-to)10036-10045
Number of pages10
JournalJournal of Physical Chemistry B
Issue number43
StatePublished - Nov 2 2017

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry


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