TABLE 3.

Characterization of wild-type TriA, AtzA, and the most improved AtzA variants derived by limited site saturation mutagenesis^a

Variant or position of variation in AtzA	Amino acid at position:					Km (μM)	kcat (s−1)	kcat/Km (s−1·M−1)	Hill coefficientb
	216	217	219	220	250
TriA	A	I	P	A	D	305	4.1	1.3 × 104	3.8
AtzAc	A	T	T	A	D	>153	2.2	1.5 × 104	ND
357	S	A	P	F	G	67	2.3	3.4 × 104	3.6
288	A	D	E	A	D	49	4.3	8.8 × 104	2.8
305	G	D	A	V	W	95	6.2	6.5 × 104	2.5
422	Y	D	Y	H	V	92	6.5	7.1 × 104	3.5
297	S	D	V	H	G	92	6.8	7.4 × 104	3.3
662	H	A	E	S	S	100	7.4	7.4 × 104	3.4
431	A	S	H	G	Y	76	8.0	1.1 × 105	3.8
841	S	D	G	S	D	105	8.5	8.1 × 104	3.8
430	G	D	G	H	G	90	12.7	1.4 × 105	3.8
734	G	D	G	H	D	62	15.1	2.4 × 105	3.6
Consensus	Y	D	T	H	E	93	27.9	3.0 × 105	3.2

^aKinetic data for atrazine dechlorination by AtzA and the AtzA variants were obtained using 4.6 to 153 μM atrazine and 10 nM enzyme. Kinetic data for melamine deamination by TriA were obtained using 80 to 560 μM melamine and 10 nM TriA. Values for the K_m, the k_cat, and the Hill coefficient for each enzyme varied by less than 10% in replicate experiments.

^bND, not determined.

^cAs the K_m of wild-type AtzA for atrazine exceeded the water solubility of atrazine, it was not possible to directly determine the K_m, k_cat, or Hill coefficient for wild-type AtzA; the maximum rate of wild-type AtzA, normalized for enzyme concentration, was used in place of k_cat.