Strong H-bonding and tritium transfer in the mechanism of triosephosphate isomerase (TIM)

Thomas K Harris, A. S. Mildvan

Research output: Contribution to journalArticle

Abstract

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 languageEnglish
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998
Externally publishedYes

Fingerprint

Dihydroxyacetone Phosphate
Triose-Phosphate Isomerase
triose-phosphate isomerase
hydrogen bonding
tritium
Tritium
phosphates
glyceraldehyde 3-phosphate
Glyceraldehyde 3-Phosphate
Biochemistry
biochemistry
protons
Protons
Proton transfer
Catalysis
catalytic activity

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Strong H-bonding and tritium transfer in the mechanism of triosephosphate isomerase (TIM). / Harris, Thomas K; Mildvan, A. S.

In: FASEB Journal, Vol. 12, No. 8, 01.12.1998.

Research output: Contribution to journalArticle

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abstract = "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.",
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