Triose phosphate isomerase template

Glu165 removes the pro-R proton from C1 of dihydroxyacetone phosphate, His95 protonates the carbonyl oxygen to give the cis-enediol, His95 then takes the O1 proton back and Glu165 delivers a proton to C2, giving D-glyceraldehyde 3-phosphate.

Mechanism step by step

Glu165 moves a proton from C1 to C2 while His95 shuttles one between the oxygens, through an enediol intermediate

1. Glu165 removes H from C1 – An enediolate forms; the O⁻ is held by Lys12 and His95
2. His95 protonates O2 – Neutral His95 acts as the acid: the cis-enediol and an imidazolate
3. His95 takes the O1 proton – The other enediolate
4. Glu165 protonates C2 – C=O forms at C1: D-glyceraldehyde 3-phosphate

Key points

• TPI is a "perfect" enzyme: kcat/KM ≈ 10⁸ M⁻¹ s⁻¹, so the rate is limited by diffusion (Albery & Knowles 1977).
• Equilibrium favours DHAP (about 96:4), but aldolase, then GAP dehydrogenase, keeps pulling GAP away, so both trioses go down glycolysis.
• A loop (residues 166–176) closes over the phosphate and keeps the enediol from losing phosphate to give toxic methylglyoxal.
• Phosphoglycolohydroxamate and 2-phosphoglycolate, enediol(ate) analogues, are tight-binding transition-state-analogue inhibitors.

Drawn with 144 atoms, 15 arrows, 8 shapes – every one editable in MolDraw.

Sources

• Nelson & Cox, Lehninger Principles of Biochemistry, 8th ed., 2021, ch. 14 Textbook
• Berg, Tymoczko, Gatto & Stryer, Biochemistry, 9th ed., 2019, ch. 16 Textbook
• Albery & Knowles, Angew. Chem. Int. Ed. Engl. 1977, 16, 285 Original paper