Energetics of triosephosphate isomerase: deuterium isotope effects in the enzyme-catalyzed reaction

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Abstract
The effect of isotopic substitution of the specifically labilized H in the substrates of triosephosphate isomerase on the steady-state rates of the enzyme-catalyzed reaction was examined. The kcat value for the enzyme-catalyzed transformation of [1(R)-2H]dihydroxyacetone phosphate is 2.9 times smaller than that for the 1(R)-1H compound. Because of the rapid loss of 2H to solvent from the enzyme-enediol complex, this factor represents the full kinetic isotope effect of the H+ abstraction step. The values of kcat and of Km for D-[2-2H] glyceraldehyde 3-phosphate are indistinguishable from those of the 2-1H material. This arises from the rapid loss of 2H from the enzyme-enediol intermediate, which results in 1H rather than 2H transfer in the rate-limiting step. The steady-state kinetic results reported in this paper qualitatively confirm and quantitatively extend the results from the previous papers on the variation of the free energy along the reaction path.