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., COHED) from Escherichia coil C catalyze two successive reactions in the homoprotocatechuate 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 completed. It has been determined that the product of CHMI, 2-oxo-5-(carboxymethyl)-3-hexenedioate (2), has the R configuration at C-5. In addition, these studies show that the enzymatic decarboxylation of 2 by COHED 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 findings, it can be concluded that the loss of carbon dioxide and the incorporation of a deuteron occur on the same side of the dienol intermediate. These results are consistent with the working hypotheses for the mechanisms of both enzymes and indicate that the sequence of events catalyzed by these two enzymes parallel the reactions catalyzed by 4-oxalocrotonate tautomerase and 4-oxalocrotonate decarboxylase in the catechol meta-fission pathway.