. 1998 Sep;64(9):3290–3299. doi: 10.1128/aem.64.9.3290-3299.1998

TABLE 3.

Kinetic constants of P. putida muconate cycloisomerase and variants with various substrates^a

Substrate	Kinetic constant of the following enzyme:
	Wild type			I54V			Y59W			A271S			K276N			F329I			L333V			I54V-F329I
	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m(min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)	K_m (μM)	k_cat (min⁻¹)	k_cat/K_m (min⁻¹ μM⁻¹)
cis,cis-Muconate	40 ± 4	12,600 ± 400	310	96 ± 8	2,500 ± 100	26	47 ± 3	1,100 ± 100	24	35 ± 3	7,100 ± 200	200	74 ± 5	2,100 ± 100	29	47 ± 4	2,600 ± 100	54	52 ± 8	68 ± 4	1.3	140 ± 10	540 ± 30	3.8
2-Chloro-cis,cis-muconate	59 ± 5	49 ± 2	0.83	47 ± 4	35 ± 1	0.75	17 ± 1	15 ± 1	0.87	60 ± 9	15 ± 1	0.25	34 ± 1	3.2 ± 0.1	0.095	37 ± 2	48 ± 1	1.3	36 ± 2	0.73 ± 0.02	0.020	33 ± 2	10 ± 0.2	0.32
3-Chloro-cis,cis-muconate^b	320 ± 200	64 ± 30	0.20	45 ± 8	200 ± 10	4.4	ND	ND	ND	180 ± 20	960 ± 60	5.4	550 ± 230	380 ± 130	0.69	690 ± 400	460 ± 230	0.67	76 ± 15	52 ± 5	0.68	210 ± 40	110 ± 10	0.54
2,4-Dichloro-cis,cis-muconate	26 ± 3	3.3 ± 0.1	0.13	93 ± 21	7.2 ± 0.8	0.078	ND	ND	ND	44 ± 8	2.9 ± 0.2	0.067	40 ± 3	0.61 ± 0.16	0.015	78 ± 16	32 ± 3	0.41	39 ± 3	0.88 ± 0.02	0.022	45 ± 6	22 ± 1	0.49
3-Fluoro-cis,cis-muconate	97 ± 21	8,900 ± 1,000	92	74 ± 8	1,200 ± 100	16	150 ± 20	3,200 ± 200	21	100 ± 12	6,000 ± 400	60	230 ± 20	5,200 ± 300	22	150 ± 10	8,000 ± 500	53	63 ± 6	4,100 ± 200	65	100 ± 9	4,900 ± 200	47
2-Methyl-cis,cis-muconate	40 ± 2	3,900 ± 100	97	58 ± 5	650 ± 30	11	69 ± 6	3,700 ± 200	53	59 ± 5	6,400 ± 200	110	180 ± 10	1,000 ± 40	5.8	68 ± 3	10,000 ± 200	150	110 ± 20	320 ± 30	2.8	57 ± 4	1,400 ± 100	24
3-Methyl-cis,cis-muconate	6.0 ± 0.5	590 ± 10	99	7.2 ± 0.7	740 ± 20	100	15 ± 1	140 ± 10	9.7	9.7 ± 0.4	680 ± 10	71	12 ± 1	95 ± 2	8.2	16 ± 1	270 ± 10	17	9.1 ± 1.7	54 ± 3	6.0	6.4 ± 0.6	310 ± 10	49

The K_m and k_cat values were determined as described in Materials and Methods. Standard deviations were calculated with the Enzfitter program. K_m values lower than and k_cat or k_cat/K_m values higher than those for the wild-type enzyme are shown in italic type and underlined if they differ by more than a factor of 2 and in boldface type and double underlined if they differ by a factor of at least 10. ND, not determined.

The reaction with 3-chloro-cis,cis-muconate was experimentally difficult to assay due to protoanemonin formation (see Materials and Methods) (4), so the standard deviations were relatively high.