Table 3.

Steady state kinetic parameters for selected pNBE variants of the DE library.

Substrate	Benzoylthiocholinea			Butyrylthiocholineb
Enzyme	kcat (1/min)	Km (mM)	kcat/Km(1/min*mM)	kcat (1/min)	Km (mM)	kcat/Km(1/min*mM)
WT	70 ± 9	1.2 ± 0.3	58 ± 16	130 ± 10	5.4 ± 0.8	24 ± 4
A107H	13 ± 1	0.6 ± 0.2	22 ± 7	35 ± 8	17 ± 5	2.0 ± 0.9
A107H Ω Loop	8 ± 1	0.9 ± 0.3	9 ± 3	10.4 ± 0.9	8.0 ± 0.7	1.3 ± 0.2
A107K	570 ± 50	1.4 ± 0.2	410 ± 70	>20	>8c	–
A107Q	40 ± 4	1.0 ± 0.2	39 ± 9	40 ± 10	19 ± 7	2 ± 1
A107R	90 ± 20	5 ± 1	20 ± 6	>50	>8c	–
A107S	39 ± 9	1.4 ± 0.6	30 ± 10	780 ± 30	14.4 ± 0.7	54 ± 3
A107T	36 ± 3	0.6 ± 0.2	60 ± 20	240 ± 30	11 ± 2	22 ± 5
A107V	38 ± 4	0.5 ± 0.2	80 ± 30	56 ± 8	8 ± 2	7 ± 2
A107Y	21 ± 2	0.6 ± 0.1	35 ± 8	45 ± 5	6.0 ± 0.9	7 ± 1
A107H/A190G	29 ± 4	0.9 ± 0.3	30 ± 10	50 ± 30	11 ± 7	5 ± 4
A107H/A190R	12 ± 1	0.6 ± 0.2	20 ± 7	200 ± 30	13 ± 2	15 ± 3
A107S/A190G	23 ± 4	2.2 ± 0.6	10 ± 3	90 ± 30	11 ± 4	9 ± 4
A107V/A190G	21 ± 2	0.6 ± 0.1	35 ± 7	45 ± 5	6.0 ± 0.9	8 ± 1
A107H/A400D	80 ± 10	2.1 ± 0.6	40 ± 10	190 ± 60	11 ± 5	18 ± 9
A107H/A190S/A400S	6.4 ± 0.9	0.8 ± 0.2	9 ± 3	115 ± 14	9 ± 1	13 ± 3

Benzoylthiocholine and butyrylthiocholine were used as substrates. Specific activities of the other variants are shown graphically in the Supplemental Information.

^aBenzoylthiocholine has limited solubility in DMSO, the highest substrate concentration tested was 2.5 mM.

^bButyrylthiocholine was also a poor substrate of pNBE, and K_m values were in the mid-millimolar range. Saturation was not achieved at the highest substrate concentration tested (8 mM). K_m values were extrapolated from double reciprocal plots.

^cSaturation was not achieved at [S] = 8 mM, and the plot of velocity vs. [S] was linear. Extrapolated K_m's exceeded 40 mM.