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 cy cle, 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 tran sformation of (E)-2-oxo-4-hexenedioate (3) to 2 through the intermedia cy of 1. Isomerases can proceed by either a ''one-base'' or a ''two-ba se'' mechanism. The overall stereochemical course of an isomerase reac tion can be used to distinguish between these two mechanisms. The ster eochemical analysis of the 4-OT reaction presents a challenge because the proposed substrate, 3, cannot be synthesized or isolated. This com plication is circumvented by utilizing strategies based on the expecte d stereospecific partitioning of 1 and related dienols in (H2O)-H-2. I t was previously determined that 4-OT ketonizes 1 to (5S)-[5-H-2]2. Be cause it was not possible to obtain sufficient quantities of [3-H-2]3 for stereochemical analysis, an alternate substrate for 4-OT, 2-hydrox y-2,4-pentadienoate (4), was used in order to determine the stereochem istry of deuterium incorporation at the 3-position. The dienol 4 is ke tonized rapidly by 4-OT to the beta,gamma-unsaturated ketone 2-oxo-4-p entenoate (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-catal yzed 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 spectroscop y. 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 s uprafacial process suggesting that 4-OT proceeds by a one-base mechani sm.