Proton transfer in the mechanism of triosephosphate isomerase

Triosephosphate isomerase (TIM) catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), wi th Glu-165 removing the pro-R proton from C1 of DHAP and neutral His-95 pol arizing the carbonyl group of the substrate. During the TIM reaction, simil ar to 2% of the pro-R tritium from C1 of DHAP is conserved and appears at C 2 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 so lvent from Glu-165 at the intermediate state and the remaining 2% is transf erred by Glu-165 to C2 of the same substrate molecule. This intramolecular transfer of tritium is therefore predicted to be independent of DHAP concen tration. On the basis of NMR detection of a strong hydrogen bond between Gl u-165 and the I-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 TPA in which Glu-165 transf ers a proton from C1 of DHAP to C2 of the enediol, and subsequently from O1 of the enediol to C2 of the product GAP. Since the pro-R proton is transfe rred to O2 instead of C2 in the criss-cross mechanism, no intramolecular tr ansfer of label from substrate to product would be expected to occur. Howev er 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 tur novers. The extent of intermolecular tritium transfer in the criss-cross me chanism would be predicted to be dependent on DHAP concentration. The exten t of tritium transfer was studied as a function of initial DHAP concentrati on 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 mh DHAP, the extent of transfer sligh tly decreased to 1.68 +/- 0.17%. In a direct test for intermolecular isotop e transfer, doubly labeled [1(R)-D,C-13(3)]DHAP and C-13-depleted [1(R)-H,C -12(3)]-DHAP were synthesized, mixed in equal amounts, and incubated at 1 m M total DHAP with TIM, GAP dehydrogenase, NAD(+), and arsenate until 50% co nversion to 3-phosphoglycerate occurred. Electrospray ionization mass spect ral analysis of the stable 3-phosphoglycerate product detected an extent of 1.4 +/- 0.4% of intramolecular D transfer from [C-13(3)]DHAP to the C-13(3 ) product, but no intermolecular transfer (less than or equal to 0.02%) of D from [C-13(3)]DHAP to the C-12(3) product. Hence, the entire transfer of hydrogen from substrate to product is intramolecular, providing no direct s upport for the criss-cross mechanism in wild-type TIM. The increase in the extent of intramolecular isotopic transfer with increasing initial DHAP con centration indicates site-site interaction in this dimeric enzyme which eit her (i) slows proton exchange with solvent from Glu-165 at the intermediate state in the classical mechanism or (ii) alters the partitioning of the ab stracted proton between transfer to C2 by the classical mechanism or to O2 by the criss-cross mechanism in which no intermolecular transfer of label o ccurs.