TABLE 1.

PA3944 wild‐type and mutant enzyme kinetic parameters toward polymyxin B, NANMO, and aspartame.

Enzyme	Polymyxin B			NANMO			Aspartame
	K m (mM)	k cat (s−1)	k cat/K m (M−1 s−1)	K m (mM)	k cat (s−1)	k cat/K m (M−1 s−1)	K m (mM)	k cat (s−1)	k cat/K m (M−1 s−1)
WT a	1.68 ± 0.07	0.51	3.0 × 102	1.07 ± 0.03	0.41	3.8 × 102	1.68 ± 0.07	0.61	3.6 × 102
R59A	0.708 ± 0.058	0.44	6.2 × 102	1.34 ± 0.09	0.39	2.9 × 102	2.00 ± 0.15	0.32	1.5 × 102
E102A a	1.05 ± 0.05	0.06	5.7 × 101	0.101 ± 0.013	0.15	1.5 × 103	0.346 ± 0.050	0.09	2.6 × 102
R106A b	0.107 ± 0.007	0.11	1.0 × 103	0.083 ± 0.007	0.16	1.9 × 103	7.74 ± 0.53	0.06	7.8 × 100
T141A	2.12 ± 0.18	0.53	2.5 × 102	1.40 ± 0.09	0.32	2.3 × 102	4.17 ± 0.20	0.19	4.3 × 101
H167A	0.122 ± 0.010	0.21	1.7 × 103	0.323 ± 0.027	0.14	4.3 × 102	3.38 ± 0.23	0.48	1.4 × 102
L169A	0.332 ± 0.039	0.42	1.3 × 103	0.871 ± 0.047	0.37	4.3 × 102	1.83 ± 0.12	0.65	3.5 × 102
H179A	0.250 ± 0.048	0.20	8.0 × 102	0.329 ± 0.035	0.27	8.2 × 102	1.64 ± 0.10	0.34	1.9 × 102

^a Data for WT and E102A toward polymyxin B and NANMO were previously reported (Baumgartner et al., 2021).

^b Significant substrate inhibition of the R106A mutant toward NANMO was observed and data were fitted to the biphasic Hill (Bi‐Hill) equation $y=\frac{P_{\mathrm{m}}}{\left[1+{\left(\frac{K_{\mathrm{a}}}{x}\right)}^{H_{\mathrm{a}}}\right]\left[1+{\left(\frac{x}{K_{\mathrm{i}}}\right)}^{H_{\mathrm{i}}}\right]}$ where y is velocity, x is concentration of substrate, P _m is the maximal velocity, K _a is the concentration of substrate at half the maximal activity for activation (reported as K _m in the table), K _i is the concentration of substrate at half the maximal activity for inhibition, H _a is the Hill coefficient for activation, and H _i is the Hill coefficient for inhibition. K _i was calculated as 0.332 ± 0.066 mM, H _a as 2.02 ± 0.33, and H _i as 0.47 ± 0.06 from the fitting.