Supplementary Information
Catalytic versatility and backups in enzyme active sites: The case of serum
paraoxonase 1
Moshe Ben-David1, Mikael Elias2, Jean-Jacques Filippi4, Elisabet Duñach4, Israel Silman3,
Joel L. Sussman1 and Dan S. Tawfik2
1
Figure 1. Overlays of the original rePON1 structure obtained at pH 4.5 (1V04, orange) and of
the new 2Å structure obtained at pH 6.5 (light blue). (A) Backbone representation of the activesite region (the catalytic calcium is shown as a green sphere, and the bound phosphate anion in
sticks). (B) The observed changes in side-chain rotamers of residues 346-348 at the active site
second-shell.
2
Figure 2. Chemical structure of substrates and their putative TS analogs used in this study.
3
Tables 1-3. Kinetic parameters of the alanine mutants of the active-site loop residues.
Table 1. Phosphotriesterase activity measured with paraoxon
Protein
K70A
Y71A
P72A
G73A
I74A
M75A
S76A
F77A
D78A
P79A
D80A
K81A
rePON1
KM (mM)
2.96±0.2
9
1.91±0.1
2
1.52±0.1
0
2.46±0.1
2
4.88±0.4
0
>5a
2.35±0.1
3
>5a
1.86±0.1
4
2.11±0.2
1
2.12±0.1
4
2.12±0.1
4
2.3±0.3
kcat (s-1)
kcat/KM (s-1M-1)
rePON1(kcat/KM)
/mutant(kcat/KM)
3.75±0.71
1264±123
1.7±0.5
0.24±0.01
124±8
17.4±3.4
1.69±0.36
1110±69
1.9±0.6
0.86±0.09
347±17
6.2±1.3
0.40±0.03
---
83±7
370±35b
25.8±5.2
5.8±1.1
3.21±0.05
---
1369±74
370±54b
1.6±0.3
5.8±1.2
3.40±0.51
1829142
1.2±0.3
2.62±0.72
1241±126
1.7±0.6
2.48±0.38
1169±78
1.8±0.5
4.94±0.24
5.01±0.59
3136±124
2149±301
0.7±0.1
1.0
a
Because of limited substrate solubility, the reaction rates did not show saturation and only an upper limit for the KM value was
provided based on the maximal substrate concentration used.
b Catalytic efficiency was obtained by linear fit of initial velocity to substrate concentration.
Table 2. Lactonase activity measured with TBBL
Protein
KM (mM)
kcat (s-1)
kcat/KM (s-1M-1)
rePON1(kcat/KM)
/mutant(kcat/KM)
K70A
Y71A
P72A
G73A
I74A
M75A
S76A
F77A
1.94±0.41
4.87±1.18
1.23±0.39
1.82±0.16
1.69±0.17
1.76±0.13
0.70±0.09
2.48±0.18
161.57±7.62
40.95±6.42
154.33±3.34
79.44±3.62
251.71±0.89
165.19±6.98
149.37±9.68
165.66±6.42
83370±17704
8411±2045
125535±39740
43599±3722
149265±14843
93753±6712
212718±28763
66763±4830
2.0±0.5
20.1±6.4
1.3±0.5
3.9±0.6
1.1±0.2
1.8±0.3
0.8±0.2
2.5±0.4
4
D78A
P79A
D80A
K81A
rePON1
0.72±0.05 88.99±2.95
1.23±0.15 136.91±7.76
0.96±0.06 133.85±4.35
0.93±0.12 167.81±13.34
1.12±0.12 188.61±15.89
123849±8703
111184±13611
139082±8919
180912±23748
169104±17947
1.4±0.2
1.5±0.3
1.2±0.2
0.9±0.2
1.0
Table 3. Aryl-esterase activity measured with phenyl acetate
Protein
K70A
Y71A
P72A
G73A
I74A
M75A
S76A
F77A
D78A
P79A
D80A
K81A
rePON1
KM (mM)
>5 a
2.52±0.19
2.80±0.20
4.39±0.36
>5 a
>5 a
2.65±0.12
>5 a
1.07±0.08
0.57±0.16
3.65±0.16
1.72±0.07
2.66±0.26
kcat (s-1)
--73.9±1.7
689.6±28.3
328.1±5
----354.1±11.1
--397.2±13
525.6±3.4
572±18.8
718.6±9.8
927±17.8
kcat/KM (s-1M-1)
28872±396 b
29398±1615
246410±6286
75064±5299
15642±315 b
69084±1693b
133466±2093
45785±1389 b
370537±15113
147393±6118
156645±3049
419303±10774
350576±33250
rePON1(kcat/KM)
/mutant(kcat/KM)
12
12
1.5
4.5
22.4
5
2.6
7.6
1
2.4
2.3
0.8
1
5
Figure 3. Docking models of rePON1 with the inhibitor 2HQ.
(A) Docking model of 2HQ (cyan) within the close structure of rePON1 (the structure the 2HQrePON1 was used as template after removing 2HQ). (B) Superposition of the docking model
within the close structure (cyan) and the actual structure of rePON1 with 2HQ (green olive). C.
Superposition of the docking model within the open structure at pH 6.5 (dark blue) and the
crystal structure of rePON1 bound with 2HQ (green olive).
6
Figure 4. Docking models of rePON1 with S-dodecanoic--lactone, and the corresponding
oxyanionic reaction intermediate, within the close structure (the rePON1-2HQ complex, with
2HQ removed).
(A) The model for S-dodecanoic--lactone. (B) Superposition of the model of S-dodecanoic-lactone (cyan) with 2HQ from the actual crystal structure (olive green). (C) Model of the
oxyanionic tetrahedral reaction intermediate of S-dodecanoic-lactone. (D) Superposition of the
oxyanionic reaction intermediates of phenylacetate (pink) and S-dodecanoic--lactone (dark
blue).
7
Figure 5.
Docking models of rePON1 with S-dodecanoic--lactone, and the
corresponding reaction intermediate, within the close structure (the rePON1-2HQ
complex with 2HQ removed). (As Fig. S6 above, but with -instead of -lactone).
(A) The model for S-dodecanoic--lactone. (B) Superposition of the model of Sdodecanoic--lactone (cyan) with 2HQ from the actual crystal structure (olive green). (C)
Model of the oxyanionic tetrahedral reaction intermediate of S-dodecanoic-lactone.
(D) Superposition of the oxyanionic reaction intermediates of S-dodecanoic-γ-lactone
(pink) and S-dodecanoic--lactone (dark blue).
8
.
Figure 6. Docking models of rePON1 with R and S isomers of thio--lactone substrates,
and of the corresponding reaction intermediates, within the close structure (the rePON12HQ complex, with 2HQ removed).
(A) The model for the R isomer. (B) Docking of the oxyanionic tetrahedral reaction
intermediate of the R isomer. (C) Docking model for the S isomer. (D) Docking of the
oxyanionic tetrahedral reaction intermediate of the S isomer.
9
Figure 7. Docking models of rePON1 with phenylacetate and of the corresponding
reaction intermediates, within the close structure (the rePON1-2HQ complex, with 2HQ
removed).
(A) The model for phenylacetate. (B) Superposition of the phenylacetate model
(magenta) with 2HQ taken from the same structure (pink). (C) Superposition of the
model of the oxyanionic intermediate state of phenyl acetate (pink) and the model of
paraoxon (ground state, in yellow) in the open structure (pH6.5). (D) Superposition of the
substituted aryl-ester 4AAP molecule (4-acetoxy-acetophenone; pink) with the model for
phenylacetate (dark blue). The active site volume cavity is shown as a light grey surface.
(E) Superposition of the docking model of 4AAP (pink) with the model of phenylacetate
(magenta).
10
Figure 8. Randomly chosen models from the docking attempts of paraoxon within the
close active-site configuration. Four random orientations of paraoxon (blue sticks) are
shown (A-D). The surface of the active-site cavity of the close configuration is shown in
transparent white.
11
12
Figure 9. Docking models of rePON1 with paraoxon and its oxyanionic reaction
intermediate.
(A) Docking model of paraoxon within the open conformation. (B) Superposition of
paraoxon (ground state, yellow) and its pentacoordinated oxyanionic hydrolytic
intermediate (yellow-orange) within the open conformation. (C) Docking model of
paraoxon within the open structure of PON1 (as in A) with the active site cavity of the
close configuration shown as a light grey surface. (D) Superposition of paraoxon with
2HQ from the close crystal structure of rePON1 (olive green). (E) Docking model of
paraoxon obtained with the close structure of rePON1 while allowing the side-chains of
Y71 and I74 to move freely during the docking simulations. The conformations of these
residues as seen in the crystal structure are shown as green sticks, and the computed as
pink sticks. (F) Docking model of the pentacoordinated oxyanionic hydrolysis
intermediate of paraoxon obtained as in part E. The original conformations of Y71 and
I74 are shown as green sticks, and the calculated as pink sticks.
13
Figure 10. Docking models of rePON1 with dihydrocoumarin and its reaction
intermediate.
(A) The model of dihydrocoumarin within the close conformation. (B) Superposition of
the docking model of dihydrocoumarin (white) with the inhibitor 2HQ (the close
conformation; olive green). (C) Superposition of the model of dihydrocoumarin (ground
state, white) and its oxyanionic tetrahedral intermediate (dark grey). (D) Superposition of
the oxyanionic intermediate of dihydrocoumarin (dark grey) and the pentacovalent
reaction intermediate of paraoxon (yellow).
14
Figure 11. Superposition of the rePON1 (apo structure at pH 6.5) and the structure of the
glucuronolactonase from Xanthomonas campestris (pdb ID: 3DR2).
(A) Superposition of the rePON1 structure (green sticks) with molecule A of the
asymmetric unit of the glucuronolactonase structure (pink sticks). Distances are given for
the calcium ion in the rePON1 structure. (B) Superposition of the rePON1 structure
(green sticks) with molecule B of the glucuronolactonase structure (pink sticks).
Distances are given for the glucuronolactonase structure. The water molecule bridging
E48 and the calcium cation is shown as a red sphere.
15
Table 4. Summary of the docking models with the 2HQ inhibitor (experimentally
measured Ki= 0.9-2.7 µM)
Enzyme
conformation
Predicted
binding energy
(kcal/mol)
Predicted Ki
(µM)
Contacting
residues
F222
close
open
-7.3
-5.9
4.7
51.9
+
+
F292
+
+
L240
+
+
I291
+
+
V346
+
+
L69
+
-
D269
+
+
N224
+
+
N168
+
+
H285
+
+
H115
+
+
E53
+
+
Y71
+
-
I74
+
-
M196
-
-
16
Table 5. Summary of the docking models with with various lactone substrates.
Substrate
S-δdecanoic
thiolactone
R- δdecanoic
thiolactone
S-δdodecanoic
lactone
Catalytic efficiency
(s-1. M-1)
8900
4100
n.d.
n.d.
n.d.
n.d.
close
close
KM
Enzyme
conformation
Liganda
Contacting residues
F222
F292
L240
I291
V346
L69
D269
N224
N168
H285
H115
E53
Y71
I74
close
S-γ-dodecanoic lactone
Dihydrocoumarin
1.23x105
(measured with
racemate)
220µM
open
close
1.19x106
129 µM
open
close
open
GS
TS
GS
TS
GS TS GS TS
GS
TS
GS
TS
GS TS GS TS
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
17
+
+
+
+
+
+
+
+
+
+
+
-
M196
- + - +
docked ligand: Substrate (GS), or oxyanionic tetrahedral reaction intermediate (TS).
-
-
-
-
a The
18
Table 6. Docking calculation summary with phenylacetate, 4AAP (aryl-esters) and
paraoxon (phosphotriester).
Activity
Substrate
Catalytic
efficiency
(s-1. M-1)
KM
Enzyme
conformation
Liganda
Contacting
residues
F222
Aryl-esterase
Phenylacetate
4AAP
Phosphotriesterase
Paraoxon
5.95x105
5.1 x103
5.8x103
1.2mM
2.6
0.8mM
close
open
close
closeb
open
GS
TS
GS
TS
GS
GS
TS
GS
TS
+
+
+
+
+
+
+
+
+
F292
+
+
+
+
+
+
+
+
+
L240
+
+
+
+
-
+
+
+
+
I291
+
+
+
+
+
+
+
+
+
V346
+
+
+
+
+
+
+
+
+
L69
-
-
-
-
+
+
+
+
+
D269
+
+
+
+
+
+
+
+
N224
+
+
-
-
+
+
+
+
N168
+
+
+
+
+
+
+
+
+
H285
+
+
+
+
+
-
-
+
+
H115
+
+
+
+
+
+
+
+
+
E53
-
+
-
+
+
-
+
-
+
Y71
+
+
-
-
+
+
+
-
-
I74
+
+
-
-
+
+
+
-
-
-
-
M196
docked ligand: Substrate (GS), or oxyanionic tetrahedral reaction intermediate (TS).
bDocked in the close structure of rePON1 with I74 and Y71 as flexible residues
a The
19