STEREOCHEMICAL STUDIES OF 5-(CARBOXYMETHYL)-2-HYDROXYMUCONATE ISOMERASE AND 5-(CARBOXYMETHYL)-2-OXO-3-HEXENE-1,6-DIOATE DECARBOXYLASE FROM ESCHERICHIA-COLI C - MECHANISTIC AND EVOLUTIONARY IMPLICATIONS

5-(Carboxymethyl)-2-hydroxymuconate isomerase (EC 5.3.2, CHMI) and 5-( carboxymethyl)-2-oxo-3-hexene-1,6-dioate decarboxylase (EC 4.1.1., COH ED) from Escherichia coil C catalyze two successive reactions in the h omoprotocatechuate meta-fission pathway resulting in the conversion of 5-(carboxymethyl)-2-hydroxymuconate (1) to 2-oxo-4-heptene-1,7-dioate (5). Stereochemical studies on both enzymatic reactions have been com pleted. It has been determined that the product of CHMI, 2-oxo-5-(carb oxymethyl)-3-hexenedioate (2), has the R configuration at C-5. In addi tion, these studies show that the enzymatic decarboxylation of 2 by CO HED generates (4Z)-2-hydroxy-2,4-heptadiene-1,7-dioate (3). Isolation and subsequent incubation of (4Z)-3 with COHED, in (H2O)-H-2, affords predominantly (3S)-[3-H-2]5. On the basis of these stereochemical find ings, it can be concluded that the loss of carbon dioxide and the inco rporation of a deuteron occur on the same side of the dienol intermedi ate. These results are consistent with the working hypotheses for the mechanisms of both enzymes and indicate that the sequence of events ca talyzed by these two enzymes parallel the reactions catalyzed by 4-oxa locrotonate tautomerase and 4-oxalocrotonate decarboxylase in the cate chol meta-fission pathway.