Table 2.

Kinetic parameters of variants at each turn of the optimization cycle of maize EPSPS with G101A as the key desensitizing mutation

For details on data collection and presentation, see the legend to Table I.

	kcat	Km PEP	kcat/Km	Ki	Km S3P	kglya	(kcat/Km) × Kib
	min−1	μm		μm		min−1
Zm native	1630 ± 14	9.5 ± 0.3	172	0.066 ± 0.003	13.2 ± 0.6	<lodc	11
G101A+3d	824 ± 15	347 ± 6.8	2.4	1430 ± 137	153 ± 3.9	17.7 ± 0.6	3,400
G101A	1000 ± 35	333 ± 26	3.0	1930 ± 40	84.0 ± 2.5	25.7 ± 0.2	5,780
H6	397 ± 4	25.1 ± 0.9	15.8	989 ± 31	14.0 ± 0.5	95.5 ± 2.0	15,700
C1	517 ± 7	18.9 ± 0.5	27.4	449 ± 48	11.4 ± 0.5	104 ± 1.6	12,300
D2	414 ± 38	14.5 ± 2.2	28.6	935 ± 66	11.3 ± 0.3	119 ± 2.0	26,700
D2–67	530 ± 17	14.4 ± 0.6	36.8	945 ± 98	10.9 ± 0.4	146 ± 2.0	34,800
D2–124	631 ± 5	18.1 ± 0.7	34.9	893 ± 53	11.1 ± 0.7	175 ± 2.5	31,130
D2c-A5	741 ± 6	18.1 ± 0.6	40.9	839 ± 40	12.6 ± 1.4	189 ± 3.1	34,350
CP4e	411 ± 5	15.5 ± 0.4	26.5	1970 ± 276	5.2 ± 0.5	176 ± 1.6	52,240
101A-97Cf	477 ± 3.5	93.0 ± 2.7	5.1	646 ± 30	36.7 ± 1.0	79.3 ± 2.2	3,300
101A-332Af	764 ± 10	123 ± 6.6	6.2	2390 ± 224	52.5 ± 3.1	47.8 ± 0.5	14,800
101A-401Gf	222 ± 2.3	291 ± 9.0	0.8	2970 ± 183	91.8 ± 4.0	23.4 ± 1.9	2,290
ACAg	408 ± 3.7	42.5 ± 1.8	9.6	1470 ± 96	31.4 ± 1.1	117 ± 1.8	14,100
A5-A101Gh	135 ± 0.8	3.9 ± 0.2	34.7	0.17 ± 0.01	11.0 ± 0.6	0.2 ± 0.08	5.9

^a Enzyme turnover (min⁻¹) at 30 μm PEP and S3P, 1 mm glyphosate (see “Results” for rational as a fitness parameter).

^b Calculated with K_m for PEP,

^c lod; limit of detection calculated as Blank + 3xSD of blank. The value is 1.5 min⁻¹.

^d Variant captured from the initial combinatorial library, having G101A plus three other mutations (E301S, E390G, V437R).

^e EPSPS from Agrobacterium sp. strain CP4, present in RoundUp Ready crops.

^f Variants of maize EPSPS having only the two mutations shown.

^g Variant having G101A, L97C, and V332A. ACA, G101A-L98C, and G101A-V332A were accessed commercially by gene synthesis.

^h D2c-A5 with Ala¹⁰¹ changed back to glycine.