Energetics of triosephosphate isomerase: the appearance of solvent tritium in substrate dihydroxyacetone phosphate and in product

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Abstract
When the isomerization of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is catalyzed by triosephosphate isomerase in 3H2O, both the substrate and product become labeled. The specific radioactivity of the product is about 80% that of the solvent, which shows that the protonation of the enediol intermediate at C-2 (to form the enzyme-bound product D-glyceraldehyde 3-phosphate) is followed by a slower step not involving H+ transfer. The specific radioactivity of the remaining substrate after partial reaction rises as the reaction proceeds and shows that the reaction intermediate that exchanges H+ with the medium returns to dihydroxyacetone phosphate (picking up 3H) about 1/3 as often as it is coverted to D-glyceraldehyde 3-phosphate. These results allow a qualitative description of the relative heights of the energy barriers in the catalyzed reaction and contribute to the quantitative analysis of the energetics of the process.