Table 1.

Kinetic parameters of InhA WT and variants

For the assays of the kinetics with octenoyl-CoA, NADH was kept constant at 300 μm, for the ones with NADH octenoyl-CoA was kept at 4 mm. All assays were measured in 30 mm PIPES, 150 mm NaCl, pH 6.8, at 30 °C at 340 nm. C2 adduct was added in powder form directly from liquid nitrogen, and its consumption was measured at 385 nm. Michaelis–Menten curves are shown in Fig. S1. NA, not applicable; enzyme catalyzes backwards reaction to substrate starting from C2-ene adduct.

Enzyme variant	Substrate	kcat,app	Km,app
		s−1	mm
InhA WT	Octenoyl-CoA	3.6 ± 0.2	0.8 ± 0.1
	NADH	3.5 ± 0.2	0.09 ± 0.01
	C2-ene adduct	3.0 ± 0.2	0.011 ± 0.002
InhA Y158F	Octenoyl-CoA	0.088 ± 0.005a	2.0 ± 0.3
	NADH	0.079 ± 0.001a	0.01 ± 0.001
	C2-ene adduct	NA	NA
InhA Y158S	Octenoyl-CoA	0.0055 ± 0.0002	0.9 ± 0.1
	NADH	0.0059 ± 0.0003	0.056 ± 0.009
	C2-ene adduct	0.23 ± 0.02	0.030 ± 0.006
InhA T196A	Octenoyl-CoA	0.050 ± 0.003	7.6 ± 0.7
	NADH	0.012 ± 0.001b	0.059 ± 0.011
	C2-ene adduct	0.077 ± 0.008	0.01 ± 0.003
InhA T196V	Octenoyl-CoA	0.0073 ± 0.0008	4.3 ± 1.0
	NADH	0.0051 ± 0.0003b	0.11 ± 0.02
	C2-ene adduct	0.056 ± 0.003	0.022 ± 0.003

^a Kinetic parameters for C2-ene adduct production. Enzyme does not catalyze full reaction.

^b NADH kinetics for T196A and T196V were not measured at octenoyl-CoA saturation because of solubility constrains leading to a lowered k_cat,app for these assays.