Supp Mat Paper 4

ESI-1
Electronic Supporting Information

Fast Enzyme Reactivity Computation. A case study of Bacillus Circulans Xylanase.
Martin R. Hediger1 , Casper Steinmann1 , Luca De Vico1 , Jan H. Jensen1
1 Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100
Copenhagen, Denmark
Corresponding Author, Email: jhjensen@chem.ku.dk
ESI-2
Figures
WT Interpolation 1 Reaction Barriers
A
WT, Interpolation 1, GNORM=1.0[kcal/(mol)]
Energy[kcal/mol]
25
CUTOFF=9
20 CUTOFF=12
CUTOFF=15
15
10
5
0
5
10
0
4
6
8
Interpolation frame
10
B
WT, Interpolation 1, GNORM=0.5[kcal/(mol)]
Energy[kcal/mol]
20
CUTOFF=9
15 CUTOFF=12
CUTOFF=15
10
5
0
5
10
15
20
0
4
6
8
Interpolation frame
10
Figure 1. wt, Interpolation 1, r =78, cutoff referring to value used in geometry optimization. All
SPE calculations done using cutoff=15
A.
A: gnorm=1.0kcal/(mol
A).
B: gnorm=0.5kcal/(mol
A).
ESI-3
Interpolation 3 Reaction Barriers

A
Q7F
V37F
Y69F
W71F
Q127F
Y166F
Energy[kcal/mol]
20
10
0
10
20
0
4
6
8
Interpolation frame
10
4
6
8
Interpolation frame
10
B
300
250
Time[h]
200
150
100
50
0
0
Figure 2. Interpolation 3 of mutants. In optimizations gnorm=0.5 kcal/(mol

A), cutoff=12
A. In
SPE calculations cutoff=15A.

A: Barriers.
B: Time requirements.
ESI-4
Reorthogonalized Barrier
[kcal/mol]
Qualitative Agreement Non-/Reorthogonalized Barriers
30
(2)
(1)
25
20
WTreortho=
13.4
15
10
5
0
WTNonreortho=
13.9
(3)
0
10
15
20
25
(4)
30
Nonreorthogonalized Barrier
[kcal/mol]
Figure 3. Dependence of reaction barriers of single mutants on reorthogonalization. Independent of

reorthogonalization, activity is qualitatively predicted the same for the datapoints within the
highlighted areas. The number of datapoints in each quadrant are 90(1), 14(2), 45(3) and 20(4),
quadrants indicated by (i) labels. Only datapoints lower than 34 kcal/mol shown.
ESI-5
Discarded mutants
Mutant
W9P
V37P
Y69P
Y80P
W71P
A115P
I118Y
Reason for discarding

Local optimization of side chain gives unphysical structure
Q7Y
V37H
Y80M
A115E
A115M
S117H
S117M
S117Q
S117R
S117W
Q127Y
Computed charge of GE structure not correct
Q7K
A115C
Q127R
Computed charge of ES wrong
Q7E
W9H
W71M
Y166R
Two atoms too close in automatically prepared structure
Table S1. Discarded single mutants.
ESI-6
Mutant
Q7W-W9E
Q7W-P116C
Q7W-F125K
W9E-Y80D
W9E-R112D
W9E-W129I
W9E-Y174D
N35E-A115I
V37T-Y65R
V37T-Y69E
V37T-I118M
Y65R-R112D
Y65R-F125K
Y65R-Q127W
Y69E-Y80D
Y69E-Y174D
W71G-Y127W
W71G-R112D
W71G-S117P
W71G-W129I
W71G-Y166V
W71G-Y174D
W71G-Y80D
Y80D-Y116V
R112D-F125K
R112D-W129I
R112D-Y166V
A115I-P116C
P116C-I118M
S117P-F125K
S117P-Y174D
W129I-Y174D
Reason for discarding

Geometry optimization time requirement too large
N35E-R112D
N35E-W129I
Y69E-S117P
W71G-P116C
S117P-Y166V
Computed charge of ES wrong
Q7W-W71G
Q7W-Y69E
R112D-P116C
R112D-Q127W
R112D-S117P
Two atoms too close in automatically prepared structure
Table S2. Discarded double mutants.
ESI-7
Single Mutant Barriers
ESI-8
Position 7
16
14 Q7A
12
10
8
6
4
2
0
0
2
16
14 Q7G
12
10
8
6
4
2
0
0
2
16
14 Q7M
12
10
8
6
4
2
0
-2
0
2
18
16 Q7S
14
12
10
8
6
4
2
0
0
2
10
18
16 Q7C
14
12
10
8
6
4
2
0
-2
0
2
10
18
16 Q7H
14
12
10
8
6
4
2
0
0
2
10
12
Q7N
10
8
6
4
2
0
-2
0
2
18
16
14
12
10
8
6
4
2
0
10
10
18
16
14
12
10
8
6
4
2
0
4
10
18
16
14
12
10
8
6
4
2
0
4
10
Q7F
120
Q7I
10
10
10
10
Q7L
100
80
60
40
20
0
10
18
16 Q7P
14
12
10
8
6
4
2
0
0
2
10
18
16 Q7V
14
12
10
8
6
4
2
0
0
2
Q7T
16
14 Q7D
12
10
8
6
4
2
0
-2
0
2
10
10
18
16 Q7R
14
12
10
8
6
4
2
0
0
2
25
Q7W
20
15
10
5
0
4
10
ESI-9
Position 9
14
W9A
12
10
8
6
4
2
0
0
2
16
14 W9F
12
10
8
6
4
2
0
0
2
16
14 W9L
12
10
8
6
4
2
0
0
2
700
W9R
600
500
400
300
200
100
0
0
2
16
14 W9Y
12
10
8
6
4
2
0
0
2
10
16
14 W9C
12
10
8
6
4
2
0
0
2
10
16
14 W9G
12
10
8
6
4
2
0
0
2
10
18
16 W9M
14
12
10
8
6
4
2
0
0
2
18
16 W9S
14
12
10
8
6
4
2
0
0
2
10
10
10
12
W9D
10
8
6
4
2
0
-2
0
2
18
16
14
12
10
8
6
4
2
0
10
10
16
14 W9E
12
10
8
6
4
2
0
0
2
10
10
18
16 W9K
14
12
10
8
6
4
2
0
0
2
10
10
16
14 W9Q
12
10
8
6
4
2
0
-2
-4
0
2
10
10
18
16 W9V
14
12
10
8
6
4
2
0
0
2
10
W9I
10
16
14 W9N
12
10
8
6
4
2
0
0
2
10
16
14 W9T
12
10
8
6
4
2
0
0
2
ESI-10
Position 35
14
12
10
8
6
4
2
0
-2
-4
-6
14
12
10
8
6
4
2
0
-2
-4
-6
N35A
0
10
10
N35F
5
0
-5
-10
-15
0
4
2
0
-2
-4
-6
-8
-10
-12
-14
10
40
35 N35Q
30
25
20
15
10
5
0
0
2
10
8
6
4
2
0
-2
-4
-6
-8
-10
10
10
10
14
12
10
8
6
4
2
0
N35D
0
15
10
10
10
10
10
N35I
-5
-10
4
10
-10
0
15
10
10
0
10
8
6
4
2
0
-2
-4
-6
-8
-10
N35R
10
10
10
10
10
N35Y
N35P
0
10
8
6
4
2
0
-2
-4
-6
-8
-10
-12
N35S
14
12
10
8
6
4
2
0
-2
-4
-6
4
0
10
8
6
4
2
0
-2
-4
-6
-8
N35M
-10
14
12 N35W
10
8
6
4
2
0
-2
-4
-6
-8
0
2
-5
5
0
-5
10
15
10
N35E
20
N35H
10
N35L
2
0
-2
-4
-6
-8
-10
-12
-14
4
14
12 N35G
10
8
6
4
2
0
-2
-4
-6
0
2
10
N35V
16
14
12
10
8
6
4
2
0
-2
N35K
18
16
14
12
10
8
6
4
2
0
-2
N35C
N35T
ESI-11
Position 37
18
16
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
V37A
0
18
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
-2
V37L
0
18
16
14
12
10
8
6
4
2
0
-2
10
12
10
8
6
4
2
0
-2
-4
-6
-8
10
10
V37Y
0
-500000
2
10
18
16 V37M
14
12
10
8
6
4
2
0
0
2
10
10
10
8 10
10
10
10
18
16 V37W
14
12
10
8
6
4
2
0
-2
0
2
10
10
10
10
V37K
0
18
16
14
12
10
8
6
4
2
0
V37T
V37E
18
16
14
12
10
8
6
4
2
0
V37N
16
14
12
10
8
6
4
2
0
4
V37I
16
14
12
10
8
6
4
2
0
-2
V37S
0
16
14
12
10
8
6
4
2
0
-2
V37G
16
14
12
10
8
6
4
2
0
-2
V37R
1e+06
500000
10
18
16
14
12
10
8
6
4
2
0
V37D
2e+06
1.5e+06
16
14
12
10
8
6
4
2
0
V37F
2.5e+06
V37C
V37Q
ESI-12
Position 65
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
Y65A
0
16
14
12
10
8
6
4
2
0
-2
10
0
15
5
0
-5
-10
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
10
10
10
10
10
10
10
16
14 Y65M
12
10
8
6
4
2
0
-2
0
2
10
10
10
18
16 Y65W
14
12
10
8
6
4
2
0
0
2
10
10
10
10
10
Y65N
0
16
14
12
10
8
6
4
2
0
Y65I
18
16
14
12
10
8
6
4
2
0
Y65R
Y65E
20
18
16
14
12
10
8
6
4
2
0
Y65H
18
16
14
12
10
8
6
4
2
0
Y65V
0
18
16
14
12
10
8
6
4
2
0
Y65Q
18
16
14
12
10
8
6
4
2
0
4
Y65L
10
Y65G
16
14
12
10
8
6
4
2
0
Y65T
0
-50
10
Y65P
50
16
14
12
10
8
6
4
2
0
Y65K
10
100
10
16
14
12
10
8
6
4
2
0
-2
Y65D
200
150
18
16
14
12
10
8
6
4
2
0
Y65F
250
Y65C
Y65S
ESI-13
Position 69
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
Y69A
0
20
18
16
14
12
10
8
6
4
2
0
10
0
16
14
12
10
8
6
4
2
0
-2
-4
90
80
70
60
50
40
30
20
10
0
18
16
14
12
10
8
6
4
2
0
-2
-10
10
10
10
10
10
10
Y69W
5
0
-5
-10
2
10
10
20
18 Y69M
16
14
12
10
8
6
4
2
0
0
2
10
10
10
10
10
10
10
Y69N
0
18
16
14
12
10
8
6
4
2
0
Y69I
100
90
80
70
60
50
40
30
20
10
0
Y69S
Y69E
16
14
12
10
8
6
4
2
0
-2
Y69H
16
14
12
10
8
6
4
2
0
-2
10
0
18
16
14
12
10
8
6
4
2
0
-2
Y69R
0
15
Y69V
Y69L
15
10
5
0
-5
-10
-15
-20
4
Y69G
10
Y69Q
0
-5
18
16
14
12
10
8
6
4
2
0
Y69K
10
18
16
14
12
10
8
6
4
2
0
Y69D
10
20
18
16
14
12
10
8
6
4
2
0
Y69F
15
Y69C
Y69T
ESI-14
Position 71
16
14 W71A
12
10
8
6
4
2
0
0
2
14
W71F
12
10
8
6
4
2
0
0
2
16
14 W71K
12
10
8
6
4
2
0
0
2
10
14
W71C
12
10
8
6
4
2
0
0
2
10
16
14 W71G
12
10
8
6
4
2
0
0
2
10
300
W71L
250
200
150
100
50
0
-50
0
2
16
14 W71S
12
10
8
6
4
2
0
0
2
18
16 W71R
14
12
10
8
6
4
2
0
-2
0
2
10
14
W71Y
12
10
8
6
4
2
0
0
2
10
10
16
14 W71D
12
10
8
6
4
2
0
-2
-4
0
2
10
16
14 W71H
12
10
8
6
4
2
0
-2
-4
0
2
10
16
14 W71N
12
10
8
6
4
2
0
-2
0
2
10
14
12 W71T
10
8
6
4
2
0
-2
-4
0
2
10
18
16 W71E
14
12
10
8
6
4
2
0
-2
0
2
10
10
10
14
12 W71Q
10
8
6
4
2
0
-2
-4
-6
0
2
10
10
14
W71V
12
10
8
6
4
2
0
0
2
10
14
12
10
8
6
4
2
0
4
10
W71I
ESI-15
Position 80
16
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
-2
Y80A
0
14
12
10
8
6
4
2
0
-2
10
0
18
16
14
12
10
8
6
4
2
0
-2
10
14
12
10
8
6
4
2
0
-2
-4
10
20
18 Y80W
16
14
12
10
8
6
4
2
0
-2
0
2
10
10
10
10
10
10
10
10
10
10
10
10
Y80Q
0
16
14
12
10
8
6
4
2
0
-2
Y80I
14
12
10
8
6
4
2
0
-2
-4
Y80T
Y80E
16
14
12
10
8
6
4
2
0
-2
Y80N
14
12
10
8
6
4
2
0
-2
4
Y80H
16
14
12
10
8
6
4
2
0
Y80S
10
18
16
14
12
10
8
6
4
2
0
-2
Y80D
14
12
10
8
6
4
2
0
-2
-4
-6
Y80L
16
14
12
10
8
6
4
2
0
-2
Y80R
10
Y80G
16
14
12
10
8
6
4
2
0
-2
Y80K
0
16
14
12
10
8
6
4
2
0
-2
Y80F
18
16
14
12
10
8
6
4
2
0
Y80C
Y80V
ESI-16
Position 112
16
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
R112A
0
16
14
12
10
8
6
4
2
0
-2
10
0
25
10
15
10
5
0
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
10
R112V
10
10
10
16
14 R112W
12
10
8
6
4
2
0
0
2
4
10
10
10
10
10
10
10
10
10
10
R112N
0
18
16
14
12
10
8
6
4
2
0
R112I
16
14
12
10
8
6
4
2
0
R112Y
10
R112S
R112E
16
14
12
10
8
6
4
2
0
16
14 R112M
12
10
8
6
4
2
0
-2
0
2
4
18
16
14
12
10
8
6
4
2
0
6
R112H
16
14
12
10
8
6
4
2
0
R112Q
10
14
12
10
8
6
4
2
0
R112D
20
18
16
14
12
10
8
6
4
2
0
R112L
16
14
12
10
8
6
4
2
0
-2
-4
R112P
10
R112G
45
40
35
30
25
20
15
10
5
0
R112K
20
0
14
12
10
8
6
4
2
0
-2
R112F
18
16
14
12
10
8
6
4
2
0
R112C
R112T
ESI-17
Position 115
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
A115D
0
18
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
10
20
18
16
14
12
10
8
6
4
2
0
10
A115V
10
10
20
18 A115W
16
14
12
10
8
6
4
2
0
0
2
4
10
10
2.5e+06
10
A115N
1.5e+06
1e+06
500000
0
2
10
10
10
A115Y
0
18
16
14
12
10
8
6
4
2
0
A115S
20
18
16
14
12
10
8
6
4
2
0
6
A115H
2e+06
10
A115L
18
16
14
12
10
8
6
4
2
0
A115R
10
18
16
14
12
10
8
6
4
2
0
A115G
14
12
10
8
6
4
2
0
-2
A115K
18
16
14
12
10
8
6
4
2
0
A115Q
0
18
16
14
12
10
8
6
4
2
0
A115I
16
14
12
10
8
6
4
2
0
A115F
8 10
A115T
10
ESI-18
Position 116
90
80
70
60
50
40
30
20
10
0
12
P116A
16
14
12
10
8
6
4
2
0
-2
P116C
10
8
6
4
2
0
0
16
14
12
10
8
6
4
2
0
10
20
18
16
14
12
10
8
6
4
2
0
-2
P116F
0
16
14
12
10
8
6
4
2
0
-2
-4
10
250
150
100
50
0
-50
0
16
14
12
10
8
6
4
2
0
-2
10
P116V
10
10
0
18
16
14
12
10
8
6
4
2
0
10
10
1e+06
500000
0
4
10
18
16
14
12
10
8
6
4
2
0
P116R
18
16 P116W
14
12
10
8
6
4
2
0
0
2
4
10
10
10
10
10
10
10
10
P116N
0
16
14
12
10
8
6
4
2
0
P116Y
8 10
P116S
14
12
10
8
6
4
2
0
-2
6
P116I
14
12
10
8
6
4
2
0
-2
P116M
1.5e+06
10
2e+06
P116E
18
16
14
12
10
8
6
4
2
0
P116H
2.5e+06
P116L
16
14
12
10
8
6
4
2
0
P116Q
200
10
P116G
16
14
12
10
8
6
4
2
0
P116K
14
12
10
8
6
4
2
0
P116D
P116T
ESI-19
Position 117
20
18
16
14
12
10
8
6
4
2
0
12
S117A
14
12
10
8
6
4
2
0
S117C
10
8
6
4
2
0
0
20
18
16
14
12
10
8
6
4
2
0
10
20
18
16
14
12
10
8
6
4
2
0
S117F
0
20
18
16
14
12
10
8
6
4
2
0
10
18
16
14
12
10
8
6
4
2
0
10
10
0
25
10
10
5
0
2
10
16
14
12
10
8
6
4
2
0
S117N
10
10
S117Y
10
10
10
10
10
S117K
0
16
14
12
10
8
6
4
2
0
S117P
S117E
16
14
12
10
8
6
4
2
0
-2
S117I
15
0
-2
-4
-6
-8
-10
-12
-14
-16
S117V
20
10
S117G
18
16
14
12
10
8
6
4
2
0
S117L
20
18
16
14
12
10
8
6
4
2
0
S117D
S117T
ESI-20
Position 118
16
14
12
10
8
6
4
2
0
-2
16
14
12
10
8
6
4
2
0
-2
I118A
0
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
-2
10
12
10
8
6
4
2
0
-2
-4
-6
40
35
30
25
20
15
10
5
0
10
10
10
10
16
14
12
10
8
6
4
2
0
-2
I118R
10
I118W
8 10
10
10
10
10
10
10
I118P
0
18
16
14
12
10
8
6
4
2
0
I118K
16
14
12
10
8
6
4
2
0
-2
I118S
10
I118N
I118E
8
6
4
2
0
-2
-4
-6
-8
-10
0
10
10
-5
-500000
8
1e+06
10
2e+06
15
500000
I118H
20
1.5e+06
16
14
12
10
8
6
4
2
0
-2
I118D
0
14
12
10
8
6
4
2
0
-2
2.5e+06
I118V
I118M
16
14
12
10
8
6
4
2
0
I118Q
10
I118G
14
12
10
8
6
4
2
0
I118L
0
16
14
12
10
8
6
4
2
0
I118F
300
250
200
150
100
50
0
-50
I118C
I118T
ESI-21
Position 125
18
16
14
12
10
8
6
4
2
0
20
18
16
14
12
10
8
6
4
2
0
F125A
0
18
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
10
18
16
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
10
F125V
10
10
10
18
16 F125W
14
12
10
8
6
4
2
0
0
2
4
10
10
25
10
10
F125Y
10
5
0
10
10
10
10
10
F125P
0
18
16
14
12
10
8
6
4
2
0
15
10
F125K
18
16
14
12
10
8
6
4
2
0
20
10
F125S
F125E
18
16
14
12
10
8
6
4
2
0
F125N
18
16
14
12
10
8
6
4
2
0
4
F125I
16
14
12
10
8
6
4
2
0
F125R
10
18
16
14
12
10
8
6
4
2
0
F125D
18
16
14
12
10
8
6
4
2
0
F125M
18
16
14
12
10
8
6
4
2
0
F125Q
10
F125H
18
16
14
12
10
8
6
4
2
0
F125L
0
16
14
12
10
8
6
4
2
0
-2
F125G
20
18
16
14
12
10
8
6
4
2
0
F125C
F125T
ESI-22
Position 127
18
16
14
12
10
8
6
4
2
0
14
12
10
8
6
4
2
0
Q127A
0
4
2
0
-2
-4
-6
-8
-10
-12
-14
-16
10
Q127F
0
20
18
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
10
6
4 Q127W
2
0
-2
-4
-6
-8
-10
-12
0
2
4
10
18
16 Q127G
14
12
10
8
6
4
2
0
0
2
4
20
15
15
10
10
10
10
10
10
15
10
5
0
2
10
14
12
10
8
6
4
2
0
Q127S
10
10
10
10
10
10
Q127N
0
18
16
14
12
10
8
6
4
2
0
Q127T
10
Q127I
16
14
12
10
8
6
4
2
0
Q127M
20
0
18
16
14
12
10
8
6
4
2
0
Q127H
0
25
Q127E
0
0
16
14
12
10
8
6
4
2
0
Q127L
18
16
14
12
10
8
6
4
2
0
Q127P
10
25
Q127D
20
18
16
14
12
10
8
6
4
2
0
Q127K
25
Q127C
Q127V
ESI-23
Position 129
16
14 W129A
12
10
8
6
4
2
0
0
2
4
16
14 W129F
12
10
8
6
4
2
0
0
2
4
10
W129K
8
6
4
2
0
-2
-4
0
2
4
16
14 W129P
12
10
8
6
4
2
0
0
2
4
14
W129T
12
10
8
6
4
2
0
0
2
4
10
16
14 W129C
12
10
8
6
4
2
0
0
2
4
10
16
14 W129G
12
10
8
6
4
2
0
0
2
4
16
14
12
10
8
6
4
2
0
10
10
18
16 W129D
14
12
10
8
6
4
2
0
0
2
4
10
16
14 W129H
12
10
8
6
4
2
0
0
2
4
10
16
14 W129M
12
10
8
6
4
2
0
0
2
4
10
20
18 W129R
16
14
12
10
8
6
4
2
0
0
2
4
10
20
18 W129Y
16
14
12
10
8
6
4
2
0
0
2
4
W129L
10
18
16 W129Q
14
12
10
8
6
4
2
0
0
2
4
10
16
14 W129V
12
10
8
6
4
2
0
0
2
4
10
16
14 W129E
12
10
8
6
4
2
0
0
2
4
10
10
10
18
16 W129N
14
12
10
8
6
4
2
0
0
2
4
10
10
18
16 W129S
14
12
10
8
6
4
2
0
0
2
4
10
10
14
12
10
8
6
4
2
0
-2
6
10
W129I
ESI-24
Position 166
18
16
14
12
10
8
6
4
2
0
14
12
10
8
6
4
2
0
-2
Y166A
0
16
14
12
10
8
6
4
2
0
10
0
16
14
12
10
8
6
4
2
0
-2
10
14
12
10
8
6
4
2
0
-2
14
12
10
8
6
4
2
0
-2
10
Y166V
10
10
50
0
-50
10
18
16 Y166W
14
12
10
8
6
4
2
0
0
2
4
10
10
10
10
10
10
10
10
10
Y166N
0
14
12
10
8
6
4
2
0
-2
Y166I
12
10
8
6
4
2
0
-2
-4
-6
Y166S
0
14
12
10
8
6
4
2
0
-2
Y166M
18
16
14
12
10
8
6
4
2
0
-2
4
Y166H
10
Y166Q
100
10
8
6
4
2
0
-2
-4
-6
4
Y166E
200
150
10
250
Y166D
18
16
14
12
10
8
6
4
2
0
-2
Y166L
12
10
8
6
4
2
0
-2
-4
Y166P
10
Y166G
14
12
10
8
6
4
2
0
-2
-4
Y166K
0
18
16
14
12
10
8
6
4
2
0
Y166F
18
16
14
12
10
8
6
4
2
0
Y166C
Y166T
ESI-25
Position 174
18
16
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
Y174A
0
18
16
14
12
10
8
6
4
2
0
10
0
18
16
14
12
10
8
6
4
2
0
10
20
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
10
10
10
10
10
10
10
10
10
18
16 Y174W
14
12
10
8
6
4
2
0
0
2
4
10
10
10
10
10
Y174N
0
18
16
14
12
10
8
6
4
2
0
-2
-4
Y174I
18
16
14
12
10
8
6
4
2
0
Y174R
Y174E
18
16
14
12
10
8
6
4
2
0
Y174M
20
18
16
14
12
10
8
6
4
2
0
4
Y174H
18
16
14
12
10
8
6
4
2
0
Y174V
10
14
12
10
8
6
4
2
0
-2
-4
Y174D
18
16
14
12
10
8
6
4
2
0
Y174Q
16
14
12
10
8
6
4
2
0
Y174T
10
Y174L
18
16
14
12
10
8
6
4
2
0
-2
-4
Y174P
10
Y174G
20
18
16
14
12
10
8
6
4
2
0
Y174K
0
18
16
14
12
10
8
6
4
2
0
Y174F
18
16
14
12
10
8
6
4
2
0
Y174C
Y174S
ESI-26
Double Mutant Barriers
ESI-27
Position 7
25
8
6
4
2
0
-2
-4
-6
-8
-10
-12
Q7W-A115I
20
15
10
5
0
0
20
18 Q7W-R112D
16
14
12
10
8
6
4
2
0
0
2
4
6
16
14 Q7W-Y166V
12
10
8
6
4
2
0
0
2
4
6
10
10
Q7W-I118M
18
16 Q7W-S117P
14
12
10
8
6
4
2
0
0
2
4
6
25
10
25
0
8
10
5
0
4
10
20
18 Q7W-Y65R
16
14
12
10
8
6
4
2
0
0
2
4
12
10 Q7W-Q127W
8
6
4
2
0
-2
-4
-6
-8
0
2
4
6
18
16
14
12
10
8
6
4
2
0
Q7W-V37T
10
10
10
Q7W-Y174D
15
15
10
20
20
18
16 Q7W-N35E
14
12
10
8
6
4
2
0
0
2
4
10
10
10
10
Q7W-W129I
0
25
Q7W-Y80D
20
15
10
5
0
6
10
ESI-28
Position 9
20
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
W9E-A115I
2.5e+06
10
W9E-P116C
2e+06
1.5e+06
1e+06
500000
0
-500000
0
16
14 W9E-W71G
12
10
8
6
4
2
0
0
2
4
8 10
10
8
6 W9E-Q127W
4
2
0
-2
-4
-6
-8
-10
0
2
4
6
18
16
14
12
10
8
6
4
2
0
14
12
10
8
6
4
2
0
W9E-F125K
10
0
15
10
5
0
-5
-10
8
10
10
18
16 W9E-Y65R
14
12
10
8
6
4
2
0
0
2
4
18
16 W9E-N35E
14
12
10
8
6
4
2
0
0
2
4
16
14
12
10
8
6
4
2
0
W9E-S117P
10
W9E-Y166V
W9E-I118M
10
10
10
10
10
W9E-V37T
0
20
18
16
14
12
10
8
6
4
2
0
W9E-Y69E
ESI-29
Position 35
6
4
2
0
-2
-4
-6
-8
14
12
10
8
6
4
2
0
N35E-F125K
25
10
15
10
5
0
-5
0
25
0
16
14
12
10
8
6
4
2
0
N35E-S117P
20
10
10
N35E-V37T
0
25
N35E-Y174D
14
12
10
8
6
4
2
0
N35E-I118M
10
N35E-P116C
14
N35E-W71G
12
10
8
6
4
2
0
0
2
4
6
30
N35E-Y65R
20
15
15
10
10
10
12
10
10
10
N35E-Y166V
10
8
6
4
2
0
8
10
0
25
N35E-Y69E
25
20
8
6 N35E-Q127W
4
2
0
-2
-4
-6
-8
-10
0
2
4
6
N35E-Y80D
20
20
15
15
10
10
10
10
5
0
0
10
ESI-30
Position 37
45
40
35
30
25
20
15
10
5
0
20
18
16
14
12
10
8
6
4
2
0
V37T-A115I
0
16
14
12
10
8
6
4
2
0
10
0
14
12
10
8
6
4
2
0
10
10
10
10
10
10
10
V37T-Y80D
V37T-Q127W
0
16
14
12
10
8
6
4
2
0
V37T-W129I
18
16
14
12
10
8
6
4
2
0
V37T-Y174D
10
8
6
4
2
0
-2
-4
-6
-8
-10
-12
V37T-P116C
14
12
10
8
6
4
2
0
V37T-S117P
16
14
12
10
8
6
4
2
0
V37T-Y166V
0
16
14
12
10
8
6
4
2
0
V37T-R112D
14
12
10
8
6
4
2
0
V37T-F125K
10
10
V37T-W71G
ESI-31
Position 65
14
12
10
8
6
4
2
0
16
14
12
10
8
6
4
2
0
Y65R-A115I
0
16
14
12
10
8
6
4
2
0
-2
10
Y65R-W129I
0
18
16
14
12
10
8
6
4
2
0
10
10
250
Y65R-P116C
20
200
15
150
10
100
50
0
0
18
16 Y65R-W71G
14
12
10
8
6
4
2
0
0
2
4
6
20
18
16
14
12
10
8
6
4
2
0
Y65R-Y69E
25
Y65R-I118M
10
10
8
6
4
2
0
10
10
0
20
18
16
14
12
10
8
6
4
2
0
Y65R-Y166V
10
Y65R-Y80D
0
0
12
Y65R-S117P
10
10
10
Y65R-Y174D
ESI-32
Position 69
25
25
Y69E-A115I
20
20
15
15
10
10
0
0
20
10
0
15
Y69E-Q127W
15
16
14
12
10
8
6
4
2
0
Y69E-F125K
10
10
5
0
-5
-5
-10
-10
0
25
10
10
Y69E-Y166V
20
15
10
5
0
0
10
Y69E-P116C
20
15
10
5
0
0
16
14
12
10
8
6
4
2
0
Y69E-R112D
10
25
Y69E-I118M
10
Y69E-W129I
10
10
16
14 Y69E-W71G
12
10
8
6
4
2
0
-2
0
2
4
6
10
ESI-33
Position 71
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
W71G-A115I
10
20
W71G-F125K
W71G-I118M
15
10
5
0
-5
0
10
10
ESI-34
Position 80
14
12
10
8
6
4
2
0
25
Y80D-A115I
20
18
16
14
12
10
8
6
4
2
0
Y80D-F125K
20
15
10
5
0
0
8
6 Y80D-Q127W
4
2
0
-2
-4
-6
-8
-10
0
2
4
6
25
10
18
16
14
12
10
8
6
4
2
0
10
10
Y80D-Y174D
20
15
10
5
0
0
10
10
Y80D-P116C
20
15
10
5
0
0
20
18
16
14
12
10
8
6
4
2
0
Y80D-R112D
25
Y80D-I118M
10
18
16
14
12
10
8
6
4
2
0
Y80D-S117P
10
10
10
Y80D-W129I
ESI-35
Position 112
2.5e+06
R112D-A115I
2e+06
1.5e+06
1e+06
500000
0
0
8 10
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
R112D-I118M
10
R112D-Y174D
10
ESI-36
Position 115
14
12
10
8
6
4
2
0
-2
-4
3
2
1
0
-1
-2
-3
-4
-5
A115I-F125K
0
18
16
14
12
10
8
6
4
2
0
10
16
14
12
10
8
6
4
2
0
A115I-W129I
10
12
10
8
6
4
2
0
-2
-4
-6
-8
A115I-I118M
10
0
20
18
16
14
12
10
8
6
4
2
0
A115I-Y166V
10
20
18
16
14
12
10
8
6
4
2
0
A115I-Q127W
10
10
A115I-Y174D
A115I-S117P
10
ESI-37
Position 116
25
6
4
2
0
-2
-4
-6
-8
-10
P116C-F125K
20
15
10
5
0
0
14
12
10
8
6
4
2
0
-2
-4
10
0
12
P116C-Y166V
16
14
12
10
8
6
4
2
0
P116C-Q127W
10
10
P116C-Y174D
10
8
6
4
2
0
10
12
P116C-S117P
P116C-W129I
10
8
6
4
2
0
10
10
ESI-38
Position 117
16
14
12
10
8
6
4
2
0
-2
10
8
6
4
2
0
-2
-4
-6
-8
S117P-I118M
10
16
14
12
10
8
6
4
2
0
S117P-Q127W
10
S117P-W129I
10
ESI-39
Position 118
16
14
12
10
8
6
4
2
0
8
6
4
2
0
-2
-4
-6
-8
-10
I118M-F125K
0
16
14
12
10
8
6
4
2
0
10
10
I118M-Y174D
12
I118M-Q127W
14
12
10
8
6
4
2
0
-2
I118M-W129I
10
8
6
4
2
0
10
10
I118M-Y166V
10
ESI-40
Position 125
10
8
6
4
2
0
-2
-4
-6
-8
20
18
16
14
12
10
8
6
4
2
0
F125K-Q127W
10
20
18
16
14
12
10
8
6
4
2
0
F125K-W129I
10
16
14
12
10
8
6
4
2
0
F125K-Y166V
10
F125K-Y174D
10
ESI-41
Position 127
12
10
8
6
4
2
0
-2
-4
-6
-8
-10
Q127W-W129I
6
4
2
0
-2
-4
-6
-8
-10
-12
-14
Q127W-Y166V
2
0
-2
-4
-6
-8
10
10
Q127W-Y174D
10
ESI-42
Position 129
10
8
6
4
2
0
-2
-4
W129I-Y166V
10
ESI-43
Position 166
12
10
8
6
4
2
0
-2
Y166V-Y174D
10

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ESI-1

Electronic Supporting Information

Interpolation 3 Reaction Barriers

Figure 2. Interpolation 3 of mutants. In optimizations gnorm=0.5 kcal/(mol

SPE calculations cutoff=15A.

Qualitative Agreement Non-/Reorthogonalized Barriers

Figure 3. Dependence of reaction barriers of single mutants on reorthogonalization. Independent of

Reason for discarding

Computed charge of GE structure not correct

Computed charge of ES wrong

Two atoms too close in automatically prepared structure

Table S1. Discarded single mutants.

Reason for discarding

Computed charge of ES wrong

Two atoms too close in automatically prepared structure

Table S2. Discarded double mutants.

Single Mutant Barriers

Double Mutant Barriers

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