Snapshot of a key intermediate in enzymatic thiamin catalysis: Crystal structure of the α-carbanion of (α,β-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae

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Abstract
Kinetic and spectroscopic data indicated that addition of the donor substrate hydroxypyruvate to the thiamin diphosphate (ThDP)-dependent enzyme transketolase (TK) led to the accumulation of the α-carbanion/enamine of (α,β-dihydroxyethyl) ThDP, the key reaction intermediate in enzymatic thiamin catalysis. The three-dimensional structure of this intermediate trapped in the active site of yeast TK was determined to 1.9-Å resolution by using cryocrystallography. The electron density suggests a planar α-carbanion/enamine intermediate having the E-configuration. The reaction intermediate is firmly held in place through direct hydrogen bonds to His-103 and His-481 and an indirect hydrogen bond via a water molecule to His-69. The 4-NH2 group of the amino-pyrimidine ring of ThDP is within 3 Å distance to the α-hydroxy oxygen atom of the dihydroxyethyl moiety but at an angle unfavorable for a strong hydrogen bond. No structural changes occur in TK on formation of the reaction intermediate, suggesting that the active site is poised for catalysis and conformational changes during the enzyme reaction are not very likely. The intermediate is present with high occupancy in both active sites, arguing against previous proposals of half-of-the-sites reactivity in yeast TK.