TABLE 5.

Product formation during 2-chloro-cis,cis-muconate turnover by variants of muconate cycloisomerase^a

Variant	Concn of residual substrate (mM)	Concn of the product (mM):			5-CML/2-CML ratio	trans-DL/(trans-DL + 5-CML) ratio (%)
		2-CML	5-CML	trans-DL
I54V	0.32	0.020	0.16	0.013	8.0	7.5
Y59W	0.38	0.088	0.0073	ND	0.083	ND
A271S	0.23	0.083	0.19	0.013	2.3	6.4
K276N	0.28	0.074	0.13	0.0039	1.8	2.9
F329I	0.33	0.13	0.0023	<0.001	0.018	ND
L333V	0.18	0.085	0.23	0.024	2.7	9.4
I54V-Y59W	0.32	0.0081	0.17	0.017	21	9.1
I54V-F329I	0.29	0.059	0.15	0.0078	2.5	4.9
Y54W-F329I	0.38	0.072	0.034	0.0025	0.47	6.8
I54V-Y59W-F329I	0.29	0.042	0.15	0.0081	3.6	5.1
None (wild type)	0.22	0.090	0.19	0.0045	2.1	2.3

^aThe reactions were carried out with 30 mM Tris-HCl (pH 7.0), 1 mM MnSO₄, 0.5 mM 2-chloro-cis,cis-muconate, and ca. 0.005 U of enzyme per ml (assayed with 2-chloro-cis,cis-muconate at pH 7.5) at 25°C. Concentrations were determined by HPLC analyses after a reaction time of 70 min. 2-CML, 2-chloromuconolactone; 5-CML, 5-chloromuconolactone; trans-DL, trans-dienelactone. ND, not determined.