Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase.
The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid- and solution-state NMR. The 13C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from -60 degrees C to 15 degrees C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps.
Rozovsky
Sharon
S
Department of Chemistry, Columbia University, New York, NY 10027, USA.
McDermott
Ann E
AE
D002247:Carbon Isotopes; D003080:Cold Temperature; D004099:Dihydroxyacetone Phosphate / Q000032:analysis / Q000378:metabolism*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism*; D006019:Glycolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D020125:Mutation, Missense; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013696:Temperature; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*
D002247:Carbon Isotopes; D029701:Saccharomyces cerevisiae Proteins; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; D014305:Triose-Phosphate Isomerase