Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of (R)-glyceraldehyde 3-phosphate in D₂O

The product distributions for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D₂O at pD 7.5-7.9 catalyzed by triosephosphate isomerase (TIM) from chicken and rabbit muscle were determined by ¹H NMR spectroscopy. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen (49% of the enzymatic products), [1(R)-²H]-DHAP from isomerization with incorporation of deuterium from D₂O into C-1 of DHAP (31% of the enzymatic products), and [2(R)-²H]-GAP from incorporation of deuterium from D₂O into C-2 of GAP (21% of the enzymatic products). The similar yields of [1(R)-²H]-DHAP and [2(R)-²H]-GAP from partitioning of the enzyme-bound enediol(ate) intermediate between hydron transfer to C-1 and C-2 is consistent with earlier results, which showed that there are similar barriers for conversion of this intermediate to the α-hydroxy ketone and aldehyde products (Knowles, J. R., and Albery, W. J. (1977) Acc. Chem. Res. 10, 105-111). However, the observation that the TIM-catalyzed isomerization of GAP in D₂O proceeds with 49% intramolecular transfer of the ¹H label from substrate to product DHAP stands in sharp contrast with the <6% intramolecular transfer of the ³H label from substrate to product GAP reported for the TIM-catalyzed reaction of [1(R)-³H]-DHAP in H ₂O (Herlihy, J. M., Maister, S. G., Albery, W. J., and Knowles, J. R. (1976) Biochemistry 15, 5601-5607). The data show that the hydron bound to the carboxylate side chain of Glu-165 in the TIM-enediol(ate) complex is not in chemical equilibrium with those of bulk solvent.