FIG. 6.

Overlaid HPLC chromatograms of 2-chloro-cis,cis-muconate before and after sequential incubation with chloromuconate cycloisomerase ClcB2 and with the 5-chloromuconolactone-converting enzyme ClcF. At least 0.1 U of the homogeneous enzymes was added in sequence to a 1-ml assay mixture containing 0.2 μmol of 2-chloro-cis,cis-muconate and 50 μmol of Tris-HCl buffer (pH 7.5). Samples, which were taken from this mixture before enzyme addition, 10 min after incubation with ClcB2 alone, and 10 min after further incubation with ClcF, were quenched by addition of 10% (vol/vol) ortho-phosphoric acid and subjected to reversed-phase HPLC. The retention times for the following compounds at a flow rate of 1 ml/min were as follows: 5-chloromuconolactone, 2.92 min; cis-dienelactone, 5.20 min; and 2-chloro-cis,cis-muconic acid, 7.47 min. The signal at 1.5 min refers to the injection peak.