Ketonization of 2-hydroxy-2,4-pentadienoate by 4-oxalocrotonate tautomerase: Implications for the stereochemical course and the mechanism

Article abstract of DOI:10.1021/ja00071a007

4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), an enzyme involved in the bacterial degradation of catechol to intermediates in the Krebs cycle, catalyzes the ketonization of 2-hydroxymuconate (1) to the α,β-unsaturated ketone (E)-2-oxo-3-hexenedioate (2). Kinetic studies on 4-OT suggest that the enzyme is an isomerase and catalyzes the transformation of (E)-2-oxo-4-hexenedioate (3) to 2 through the intermediacy of 1. Isomerases can proceed by either a "one-base" or a "two-base" mechanism. The overall stereochemical course of an isomerase reaction can be used to distinguish between these two mechanisms. The stereochemical analysis of the 4-OT reaction presents a challenge because the proposed substrate, 3, cannot be synthesized or isolated. This complication is circumvented by utilizing strategies based on the expected stereospecific partitioning of 1 and related dienols in ²H₂O. It was previously determined that 4-OT ketonizes 1 to (5S)-[5-²H]2. Because it was not possible to obtain sufficient quantities of [3-²H]3 for stereochemical analysis, an alternate substrate for 4-OT, 2-hydroxy-2,4-pentadienoate (4), was used in order to determine the stereochemistry of deuterium incorporation at the 3-position. The dienol 4 is ketonized rapidly by 4-OT to the β,γ-unsaturated ketone 2-oxo-4-pentenoate (5) before a much slower conversion to its α,β-isomer, 2-oxo-3-pentenoate (6). This behavior allows for the accumulation of 5 in solution. In order to assign the stereochemistry, the 4-OT-catalyzed ketonization of 4 was performed in ²H₂O. The product, [3-²H]5, was trapped with NaBH₄, processed to [3-²H]malate by chemical and enzymatic degradative procedures, and analyzed by ¹H NMR spectroscopy. It was concluded that 4-OT ketonizes 4 stereoselectively to (3R)-2-oxo-[3-²H]-4-pentenoate. This result and the previous stereochemical finding indicate that the isomerization of 3 to 2 is predominantly a suprafacial process suggesting that 4-OT proceeds by a one-base mechanism.