CHEMICAL AND ENZYMATIC KETONIZATION OF 5-(CARBOXYMETHYL)-2-HYDROXYMUCONATE

The conjugated enol, 5-(carboxymethyl)-2-hydroxymuconate (1),1 is a stable dienol generated in the course of bacterial catabolism of 4-hydroxyphenylacetate by the enzymes of the homoprotocatechuate pathway. The dienol ketonizes chemically in aqueous solution and enzymatically by the action of 5-(carboxymethyl)-2-hydroxymuconate isomerase (EC 5.3.2) to an alpha,beta-unsaturated ketone, (E)-2-oxo-5-(carboxymethyl)-3-hexenedioate (2). Mechanistic studies for both processes remain largely unexplored, An examination of the behavior of 1 in phosphate buffer has been completed using UV and H-1 NMR spectroscopy. The results of these studies indicate that a rapid equilibrium forms between 1 and the beta,gamma-unsaturated ketone, 2-oxo-5-(carboxymethyl)-4-hexenedioate (6) before a much slower conversion to 2. The spectroscopic results were confirmed by reduction of the isomeric ketones with sodium borohydride and subsequent identification of the products. The rapid interconversion of 1 and 6 in aqueous phosphate buffer raises the question of whether the enzyme has a preference for one isomer as its substrate. The values of k(cat)/K(M) determined for 1 and a mixture of 1 and 6 suggest that both 1 and 6 are excellent substrates for CHMI. In addition, these studies indicate that 1 is kinetically competent to be an intermediate in the overall reaction. A reasonable hypothesis to explain these observations is that 5-(carboxymethyl)-2-hydroxymuconate isomerase catalyzes the transformation of 6 to 2 through the intermediacy of 1. The relevance of these findings to a related dienol, 2-hydroxymuconate (3), and the in vivo catabolism of 3,4-dihydroxyphenylacetate by the enzymes of the homoprotocatechuate pathway are briefly discussed.