KETONIZATION OF 2-HYDROXY-2,4-PENTADIENOATE BY 4-OXALOCROTONATE TAUTOMERASE - IMPLICATIONS FOR THE STEREOCHEMICAL COURSE AND THE MECHANISM

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 alpha,beta-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 (H2O)-H-2. It was previously determined that 4-OT ketonizes 1 to (5S)-[5-H-2]2. Because it was not possible to obtain sufficient quantities of [3-H-2]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 beta,gamma-unsaturated ketone 2-oxo-4-pentenoate (5) before a much slower conversion to its alpha,beta-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 (H2O)-H-2. The product, [3-H-2]5, was trapped with NaBH4, processed to [3-H-2]malate by chemical and enzymatic degradative procedures, and analyzed by H-1 NMR spectroscopy. It was concluded that 4-OT ketonizes 4 stereoselectively to (3R)-2-oxo-[3-H-2]-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.