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, ~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 (≤0.02%) of D from [13C3]DHAP to the 12C3 product. Hence, the entire transfer of hydrogen from substrate to product is intramolecular, providing no direct support for the criss-cross mechanism in wild-type TIM. The increase in the extent of intramolecular isotopic transfer with increasing initial DHAP concentration indicates site-site interaction in this dimeric enzyme which either (i) slows proton exchange with solvent from Glu- 165 at the intermediate state in the classical mechanism or (ii) alters the partitioning of the abstracted proton between transfer to C2 by the classical mechanism or to O2 by the criss-cross mechanism in which no intermolecular transfer of label occurs.
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