TIM catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) during which -2% of pro-R tritium from Cl of DHAP appears at C2 of GAP [Nickbarg & Knowles (1988) Biochemistry 27, 5939]. From 'H NMR studies of strong H-bonding between TIM and an analog of the enediol(ate) intermediate, we have suggested a dual role for Glu-165 in catalysis of proton transfer, not only between the Cl and C2 carbons, but also between the Ol and O2 oxygens in the conversion of DHAP to GAP [Biochemistry 36, 14661]. This mechanism requires intermolecular tritium transfer between DHAP and GAP which should show a DHAP concentration dependence, while intramolecular tritium transfer should not. The extent of tritium transfer from DHAP, highly tritiated at the pro-R position, at 50% conversion to GAP, showed triphasic behavior as a function of DHAP concentration: Between 0.03 and 0.3 mM DHAP, a constant tritium transfer of 1.19 ± 0.03% occurred. Between 0.3 and 1.0 mM DHAP, transfer progressively increased as a function of [DHAP] to 2.17 ±0.15%. Between 1.0 and 7.0 mM DHAP, transfer slightly decreased to 1.68 ±0.17%. Phase 1 reflects intramolecular transfer between Cl and C2 of the same substrate/product molecule. Phase 2 indicates an intermolecular contribution from DHAP to GAP comparable in magnitude to the intramolecular exchange. Phase 3 reflects intermolecular back-exchange of tritium from labeled to unlabeled DHAP. The triphasic nature of the tritium exchange provides evidence for a dual mechanism for the TIM reaction in which the abstracted pro-R proton partitions between O2 and C2 of the enediol(ate) intermediate.
|Original language||English (US)|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology