TABLE 1.

Effects of mutations in TrzN on protein yield, stability, and kinetic parameters^a

Amino acidsb	Yield (mg/liter)	Tm50(app) (oC) (holo-TrzN)	kcat (s−1)	Km (μM)	kcat/Km (105 M−1 s−1)
D, L, A	<0.2	52.1	2.1	19.7	1.1
N, L, A	<0.1	ND	2.6	21.3	1.2
D, P, A	0.6	51.3	3.1	48.6	0.6
D, L, V	8.4	51.8	2.8	18.9	1.5
N, P, A	1.2	52.3	3.2	52.2	0.6
N, L, V	14.4	54.4	2.6	21.7	1.2
D, P, V	28.2	51.0	3.2	54.2	0.6
N, P, V	67.2	53.6	3.7	53.0	0.7

^aYield was measured as purified protein, stability was measured as the temperature at which 50% activity was lost (68), and kinetic parameters were measured with atrazine as the substrate using a previously published protocol (38). The T_m^50(app) values could not be determined for the majority of the apoenzyme samples because the protein immediately unfolded upon removal of the metal ion.

^bAmino acids at positions 38, 131, and 159, respectively.