Table 1.

Mutant enzymes with experimentally observed specificity (k_cat/K_m), k_cat, and K_m for a target substrate and the WT substrate (Phe)

Redesign target	Enzyme	Rank	Target substrate			WT substrate (Phe)
			kcat min−1	Km mM	kcat/Km mM−1 min−1	kcat min−1	Km mM	kcat/Km mM−1 min−1
Leu†	T278L/A301G	1	1.16 ± 0.10	0.015 ± 0.002	79.49 ± 13.67	3.37 ± 0.08	0.097 ± 0.013	34.94 ± 4.76
	T278M/A301G	8	2.63 ± 0.24	0.130 ± 0.009	20.34 ± 3.11	4.25 ± 0.16	0.318 ± 0.009	13.35 ± 0.14
	A322V/A301G	9	4.18 ± 0.52	0.448 ± 0.019	9.34 ± 1.08	3.17 ± 0.2	0.195 ± 0.019	16.35 ± 1.3
	WT‡		28.74 ± 1.58	6.98 ± 1.00	4.15 ± 0.36	1.73 ± 0.29	0.0018 ± 0.0004	951.4 ± 111.2
Arg†	T278D/A301G	1	0.238 ± 0.007	46.43 ± 4.79	0.0051 ± 0.0004	0.50 ± 0.02	0.153 ± 0.02	3.29 ± 0.38
	WT		ND§	ND	ND
Glu†	T278H/A301G	2	0.3773 ± 0.035	25.49 ± 5.65	0.0151 ± 0.0023	0.38 ± 0.03	0.027 ± 0.006	14.7 ± 2.48
	WT		ND	ND	ND
Lys†	T278D/A301G	4	1.09 ± 0.08	78.33 ± 16.39	0.014 ± 0.003	0.50 ± 0.02	0.153 ± 0.02	3.29 ± 0.38
	WT		ND	ND	ND
Asp†	T278K/A301G	3	>0.25¶			16.19 ± 1.32	28.93 ± 1.91	0.56 ± 0.06
	WT		ND	ND	ND
Leu‖	T278L/A301G/S447N		0.85 ± 0.11	0.0054 ± 0.001	159.86 ± 14.98	3.36 ± 0.23	0.168 ± 0.027	20.38 ± 3.97
	I277L/T278L/A301G		1.52 ± 0.09	0.013 ± 0.002	119.79 ± 9.75	5.21 ± 0.22	0.306 ± 0.004	17.00 ± 0.55
	V187L/T278L/A301G		1.69 ± 0.22	0.011 ± 0.001	155.51 ± 26.71	3.24 ± 0.16	0.201 ± 0.036	16.43 ± 2.47
	I277L/T278L/A301G/S447N		0.37 ± 0.04	0.0054 ± 0.0007	69.07 ± 13.09	2.09 ± 0.10	0.282 ± 0.023	7.44 ± 0.67

^†K* active-site mutants with their respective ranks from the 2-point mutation searches for the different substrates.

^‡For clarity, the WT enzyme:WT substrate rates are shown only once.

^§ND, not detectable.

^¶K_m and k_cat/K_m cannot be determined accurately because the solubility of Asp (≈50 mM in water) limits the reaction velocity under the experimental condition, in which the velocity remains linearly dependent on the concentration of the substrate.

^‖ Bolstering mutations added to the T278L/A301G mutant with Leu as substrate.