Proton transfer in the mechanism of triosephosphate isomerase

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), with Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 polarizing the carbonyl group of the substrate. During the TIM reaction, approximately 2% of the pro-R tritium from C1 of DHAP is conserved and appears at C2 of GAP [Nickbarg, E. B., and Knowles, J. R. (1988) Biochemistry 27, 5939]. In the "classical" mechanism, 98% of the pro-R tritium exchanges with solvent from Glu-165 at the intermediate state and the remaining 2% is transferred by Glu-165 to C2 of the same substrate molecule. This intramolecular transfer of tritium is therefore predicted to be independent of DHAP concentration. On the basis of NMR detection of a strong hydrogen bond between Glu-165 and the 1-OH of an analogue of the enediol intermediate [Harris, T. K., Abeygunawardana, C., and Mildvan, A. S. (1997) Biochemistry 36, 14661], we have suggested a "criss-cross" mechanism for TIM in which Glu-165 transfers a proton from C1 of DHAP to O2 of the enediol, and subsequently from O1 of the enediol to C2 of the product GAP. Since the pro-R proton is transferred to O2 instead of C2 in the criss-cross mechanism, no intramolecular transfer of label from substrate to product would be expected to occur. However, intermolecular transfer of label could occur if the label exchanges from O2 into a group on the protein and is transferred to GAP in subsequent turnovers. The extent of intermolecular tritium transfer in the criss-cross mechanism would be predicted to be dependent on DHAP concentration. The extent of tritium transfer was studied as a function of initial DHAP concentration using DHAP highly tritiated at the pro-R position. At 50% conversion to GAP, triphasic tritium transfer behavior was found. For phase 1, between 0.03 and 0.3 mM DHAP, a constant extent of tritium transfer of 1.19 +/- 0.03% occurred. For phase 2, between 0.3 and 1.0 mM DHAP, the extent of transfer progressively increased as a function of DHAP concentration to 2.17 +/- 0.15%. For phase 3, between 1.0 and 7.0 mM DHAP, the extent of transfer slightly decreased to 1.68 +/- 0.17%. In a direct test for intermolecular isotope transfer, doubly labeled [1(R)-D, 13C3]DHAP and 13C-depleted [1(R)-H,12C3]DHAP were synthesized, mixed in equal amounts, and incubated at 1 mM total DHAP with TIM, GAP dehydrogenase, NAD+, and arsenate until 50% conversion to 3-phosphoglycerate occurred. Electrospray ionization mass spectral analysis of the stable 3-phosphoglycerate product detected an extent of 1.4 +/- 0.4% of intramolecular D transfer from [13C3]DHAP to the 13C3 product, but no intermolecular transfer (