Supporting Information for:

Cooperative

Electrostatic

Interactions

Drive

Functional

Evolution in the Alkaline Phosphatase Superfamily

Alexandre Barrozo , Fernanda Duarte, Paul Bauer, Alexandra T. P. Carvalho, and Shina C. L.
Kamerlin*
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University,
BMC Box 596, S-75124 Uppsala, Sweden
Corresponding author email addresses:
kamerlin@icm.uu.se

Both authors contributed equally

S1

Table of Contents
S1.

Calibrating the Relevant Reference Reactions for the EVB Simulations

S4

S1.1.

Theoretical background

S4

S1.2.

Background reaction of phenyl p-nitrophenyl phosphonate

S6

S1.3.

Background reaction of ethyl p-nitrophenyl phosphate

S7

S1.4.

Background reaction of p-nitrophenyl phosphate and sulfate monoesters

S7

S1.5.

Background reaction of phenyl p-nitrophenyl sulfonate

S8

S2.

References

S9

S3.

Figures & Tables

S11

Figure S1: Representation of the cationic dummy model

S11

Figure S2: Time-averages of the root mean square deviation of the protein backbone

S12

atoms for the monomeric unit of the WT and manually generated mutant forms of
RlPMH with phenyl p-nitrophenyl phosphonate bound in the active site
Figure S3: Time-averages of the root mean square deviation of the protein backbone

S13

atoms for the monomeric unit of the WT and manually generated mutant forms of
RlPMH with ethyl p-nitrophenyl phosphate bound in the active site
Figure S4: pH rate profiles for the hydrolysis of the substrates of BcPMH

S14

Figure S5: Average P(S)-O and H-N/Olg distances along the reaction coordinate for

S15

the hydrolysis of the relevant group transfer reaction in the wild-type form of BcPMH
Figure S6: Average P(S)-O and H-N/Olg distances along the reaction coordinate for

S16

the hydrolysis of the relevant group transfer reaction in the K337 form of RlPMH
Table S1: Kinetic parameters for the uncatalyzed and catalyzed hydrolyses of phenyl

S17

p-nitrophenyl phosphonate (PPP), ethyl p-nitrophenyl phosphate (PET), p-nitrophenyl

sulfate (PNS), phenyl p-nitrophenyl sulfonate (PPS), and p-nitrophenyl phosphate
monoanion (PNPH)
Table S2: Kinetic parameters for the catalyzed hydrolyses of phenyl p-nitrophenyl

S18

phosphonate and ethyl p-nitrophenyl phosphate by the wild type and different mutant
forms of RlPMH
Table S3: Partial charges at the reactant state (RS) and transition state (TS) for the

S2

S19

nucleophile oxygen (Onuc), the central P/S atom plus non-bridging oxygens, and the
leaving group oxygen (Olg)
Table S4: Average P(S)-O and H-N/Olg distance at the transition state of the

S20

hydrolysis for the relevant group transfer reaction in water and in the wild-type forms
of RlPMH and BcPMH
Table S5: Average P(S)-O distance at the transition state of the hydrolysis of phenyl

S21

p-nitrophenyl phosphonate and ethyl p-nitrophenyl phosphate in the different mutant

forms of RlPMH
Table S6: Average P(S)-O and H-N/Olg distances at the transition state in water, wild-

S22

type and K337A mutant forms of RlPMH

Table S7: Volume, surface properties and number of catalytic activities of several

S23

members of the AP superfamily

Table S8: Set of parameters used for the pocket analysis of several members of the AP

S23

superfamily
Table S9: EVB mapping parameters used in this work

S24

S4.

Empirical Valence Bond Parameters Used in This Work

S24

Off-diagonal parameters and gas-phase shift values used in the present work

S24

EVB states and atom numbering order used as a reference in this work

S25

Van der Waals parameters used for atoms constituting the reacting part of the system

S27

Atom types in different VB states

S28

Atomic charges

S32

Bond parameters for covalent bonds of the reacting part of the system

S36

Angle parameters used for bending adjacent bonds in reacting part of the system

S36

Torsion parameters used in reacting part of the system

S37

S3

S1. Calibrating the Relevant Reference Reactions for the EVB Simulations
S1.1. Theoretical background
The empirical valence bond is unique in that it is a fully classical approach that uses the
mixing of classical force fields in a valence bond-based quantum mechanical framework to
describe chemical reactivity1-3. As described in the main text, one of the main advantages of this
approach is that it is very fast, which allows for extensive conformational sampling compared to
other semi-empirical or ab initio QM/MM approaches. At the same time, it carries a very large
amount of chemical information, allowing one to model bond-making and bond-breaking
processes in a chemically meaningful way3. The success of this approach as applied to numerous
biological systems has been extensively documented3-5, and its ability to predict and rationalize
mutational effects has led the EVB approach to also be suggested to be a powerful tool for
computer-guided enzyme (re-)design6-8.
Part of the EVB philosophy is based on the existence of a well-defined reference state4, which
is used for all subsequent calculations on that reaction. Once a reference state has been clearly
and rigorously defined and parametrized, the same parameter set can then be used completely
unchanged for the wild-type and all mutant forms of the enzyme (see discussion in ref. 3),
allowing one to directly examine the catalytic effect of the enzyme and compare the effect of
mutations on this effect without the need for any adjustable parameters. When calibrating the
reference state, one important aspect to take into account is that the preferred mechanism in
solution, without the catalyst present, can be different from the one in the enzyme active site, as
one feature of the enzyme may be to change the mechanism of the uncatalyzed reaction.
However, the relevant reference state in this valence bond framework is an identical reaction in
the absence of the catalyst. Where direct experimental data is not available, this energetic

S4

difference can be obtained either through high level quantum chemical calculations, through
consideration of relevant pKa differences, or through indirect considerations of analogous
experimental data, as outlined in ref. 4.
As outlined in Figure 2 of the main text, the PMHs being considered in this work catalyze
the hydrolysis of a range of phosphate, sulfate, phosphonate and sulfonate monoesters. As there
is no direct experimental data available on the reactivity of these compounds using the same
nucleophile considered in the present work (anionic acetaldehyde hydrate), we have calibrated
the energetics of our reference reactions to extrapolated experimental data. Specifically, we have
taken into account the wealth of experimental data available on both the alkaline and
spontaneous hydrolyses9-12 of the substrates studied here, adjusting for nucleophile concentration
(in the case of the alkaline hydrolysis) and nucleophile pKa in order to obtain approximate
activation barriers for our model reaction in aqueous solution and to quantify the catalytic effect
of the enzyme. As we are not taking into account the second step of the reaction in this work (i.e
the likely spontaneous hemiacetal cleavage13), the precise reaction free energy is less crucial, and
we therefore used a constant value of -8.8 kcal mol-1 as an approximation for all reactions
modeled in aqueous solution, based on the difference between the pKa of the nucleophile (13.5)14
and leaving group (7.14)9. Additionally, in the case of the hydrolysis of the phosphate and
phosphonate monoesters by RlPMH, we also calculate the activity of a series of mutant forms of
the enzyme, and the fact that we can reproduce relevant trends between mutants with high
accuracy further supports the reliability of our model. Our corresponding considerations in
calibrating the activation barriers for each substrate of interest are shown below. Taking into
account this information we have calibrated the hydrolysis steps of the reaction as described in
Sections S1.2 to S1.5.

S5

S1.2. Background reaction for the hydrolysis of phenyl p-nitrophenyl phosphonate

The most challenging of the reactions to calibrate in this study is the hydrolysis of phenyl pnitrophenyl phosponate (PPP), due to the limited experimental data available on this reaction.
That is, while there is extensive kinetic data available on the hydrolysis of methyl p-nitrophenyl
phosphonate15-17, there is much less so for its phenyl counterpart. In a recent work, McWirther
and coworkers15 obtained a rate of kOH- = 1.2 x 10-6 s-1 for the alkaline hydrolysis of the methyl
phosphonate at 25 C in 0.1 M NaOH. The rate of the same reaction at 30 C has been measured
to be 2.3 x 10-5 M-1 s-1, at 0.169 M nucleophile concentration, i.e 3.9 x 10-6 s-1 after adjusting for
nucleophile concentration17. The corresponding spontaneous reaction has a measured rate of 1.95
x 10-9 s-1 at the same temperature, corrected for the 55 M concentration of water17. Correcting
once again for the pKb of acetaldehyde hydrate (13.5), one obtains a hypothetical estimate of
~1.46 x 10-6 s-1 for anionic acetaldehyde hydrate attack on methyl p-nitrophenyl phosphonate.
Hollfelder and coworkers18 provide an estimate of kH2O = 1.71 x 10-11 s-1 for the spontaneous
hydrolysis of phenyl p-nitrophenyl phosphonate, making it almost 114 times slower than the
corresponding spontaneous hydrolysis of methyl phosphate, so we can adjust our rate by a
similar factor to obtain an approximation of 1.28 x 10-8 s-1 for the attack of anionic acetaldehyde
hydrate on the phenyl phosphonate, corresponding to an estimated activation barrier of ~28.4
kcal mol-1. In this specific case, a large number of approximations are necessary to reach this
estimate due to the lack of direct experimental data for this less studied substrate in aqueous
solution. However, fortunately, in the case of the corresponding enzyme catalyzed reaction, the
phosphonate monoester is one of the substrates for which extensive mutational data is available
in the literature19, and the fact that we can reproduce these relative mutational effects with very
high fidelity (Table 3 of the main text) compensates for the uncertainty in the precise activation

S6

barrier in aqueous solution. We also would like to emphasize that the main purpose of including
the reference reaction at all is to see whether we are able to observe any substantial barrier
reduction compared to background in our calculations for the mechanism we are modeling, and
in light of the mutational data available, we could just as easily have used the wild-type enzyme
as our reference state.
S1.3. Reference reaction of ethyl p-nitrophenyl phosphate diester
Compared to the phenyl phosphonate, calibrating the background reaction for the ethyl pnitrophenyl phosphate diester is straightforward, as there is extensive experimental data available
on the hydrolysis of this compound, and there is no need to extrapolate from different spectator
groups. Hollfelder and coworkers18 have used Purcell and Hengges published temperature
dependence12 to obtain an approximate rate constant of kH2O = 2.6 x 10-13 s-1 (corrected for 55 M
water concentration) for the hydrolysis of this compound at 30 C. The corresponding rate
constant for the alkaline hydrolysis12 of the same compound (corrected for 0.1 M of NaOH
concentration) is 8.02 x 10-8 s-1. Correcting again for the pKb of acetaldehyde hydrate gives an
approximate rate constant of 1.62 x 10-8 s-1, or an activation barrier of ~28.6 kcal mol-1. As in
the case of the phosphonate, we also have extensive mutational data for this substrate19.
S1.4. Reference reactions of the p-nitrophenyl phosphate and sulfate monoesters
Of all the compounds of interest in the present work, the p-nitrophenyl phosphate9,10,20 and
sulfate11,20 monoesters are the best studied, as they are prototypes systems for the reactivity of
phosphate and sulfate monoesters respectively. In this case we took into consideration the pnitrophenyl phosphate monoanion rather than the dianionic form, as discussed in the main text.
For the p-nitrophenyl sulfate monoester the rate constants are kOH- = 4.99 x 10-9 s-1, and kH2O =
2.47 x 10-9 s-1 at 35 C11 (all rates corrected for nucleophile concentration, however, note that

S7

kOH- could in principle correspond to C-O rather than P(S)-O cleavage, and provides therefore a
only lower limit for this rate). Adjusting for the pKb of acetaldehyde hydrate gives estimated
activation barriers of 29.8 kcal mol-1 for (anionic) acetaldehyde hydrate attack on the sulfate
monoester. For the p-nitrophenyl phosphate monoanion, Kirby and Varvoglis9 reported a value
of 25.4 kcal mol-1 for H and -4.5 e.u. for S, giving a G of 25.7 kcal mol-1 at 25 C. In
the case of the phosphate, there is no direct experimental data available for the alkaline
hydrolysis of the monoanion, as the concentration of the protonated form of the phosphate will
decrease as the concentration of hydroxide increases. However, one can, to an approximation,
assume that the rate of the corresponding methylated analogue is similar to the rate of attack on
the monoanionic phosphate (see ref. 21). In this case, a value of 70 kJ mol-1 for H and -117 J
mol-1 K-1 for S22 at 25 C (i.e. G= 25.0 kcal mol-1 at 25 C) has been reported for the
alkaline hydrolysis of ethyl p-nitrophenyl phosphate, which we use as a reference in the present
work.
S1.5. Reference reaction of phenyl p-nitrophenyl sulfonate
Although not as extensively studied as the previous compounds, there still nevertheless exists
experimental data on the hydrolysis of sulfonate esters with various leaving groups. Specifically,
Hollfelder and coworkers23 have examined the alkaline hydrolysis of a range of aryl benzene
sulfonates in 0.1 M NaOH, including the 4-nitro substituted compound, obtaining a rate constant
of 1.84 x 10-3 s-1 for this compound23, corrected for nucleophile concentration. They also provide
an estimate of kH2O = 5.5 x 10-9 s-1 at 25 C for the spontaneous hydrolysis of this compound (see
Supporting Information of ref. 18), which would suggest that the rate of hydrolysis via an alkoxy
nucleophile would be in the range of 2.51 x 10-4 s-1. This would correspond to an activation
barrier of ~22.4 kcal mol-1.

S8

S2. References

(1) Warshel, A.; Weiss, R. M. J. Am. Chem. Soc. 1980, 102, 6218-6226.
(2) Warshel, A. Computer Modeling of Chemical Reactions in Enzymes and Solutions; Wiley:
New York, 1991.
(3) Kamerlin, S. C. L.; Warshel, A. WiRE: Comp. Mol. Sci. 2011, 1, 30-45.
(4) Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, M. H. M. Chem. Rev.
2006, 106, 3210-3235.
(5) Shurki, A.; Derat, E.; Barrozo, A.; Kamerlin, S. C. L. Chem. Soc. Rev. 2015, 44, 1037-1052.
(6) Roca, M.; Vardi-Kilshtain, A.; Warshel, A. Biochemistry 2009, 48, 3046-3056.
(7) Frushicheva, M. P.; Cao, J.; Chu, Z. T.; Warshel, A. Proc. Natl. Acad. Sci. U. S. A. 2010,
107, 16869-16874.
(8) Frushicheva, M. P.; Cao, J.; Warshel, A. Biochemistry 2011, 50, 3849-3858.
(9) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415-423.
(10) Kirby, A. J.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 3209-3216.
(11) Benkovic, S. J.; Benkovic, P. A. J. Am. Chem. Soc. 1966, 88, 5504-5511.
(12) Purcell, J.; Hengge, A. C. J. Org. Chem. 2005, 70, 8437-8442.
(13) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1961, 83, 1743-1750.
(14) Bell, R. P.; McTigue, P. T. J. Chem. Soc. 1960, 2983-2994.
(15) McWhirter, C.; Lund, E. A.; Tanifum, E. A.; Feng, G.; Sheikh, Q. I.; Hengge, A. C.;
Williams, N. H. J. Am. Chem. Soc. 2008, 130, 13673-13682.
(16) Behrman, E. J.; Biallas, M. J.; Brass, H. J.; Edwards, J. O.; Isaks, M. J. Org. Chem. 1970,
35, 3063-3069.
(17) Behrman, E. J.; Biallas, M. J.; Brass, H. J.; Edwards, J. O.; Isaks, M. J. Org. Chem. 1970,
S9

35, 3069-3075.
(18) van Loo, B.; Jonas, S.; Babtie, A. C.; Benjdia, A.; Berteau, O.; Hyvnen, M.; Hollfelder, F.
Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 2740-2745.
(19) Jonas, S.; van Loo, B.; Hyvnen, M.; Hollfelder, F. J. Mol. Biol. 2008, 384, 120-136.
(20) Hoff, R. H.; Hengge, A. C. J. Org. Chem. 1998, 63, 6680-6688.
(21) Admiraal, S. J.; Herschlag, D. J. Am. Chem. Soc. 2000, 122, 2145-2148.
(22) Williams, N. H.; Cheung, W.; Chin, J. J. Am. Chem. Soc. 1998, 120, 8079-8087.
(23) Babtie, A. C.; Lima, M. F.; Kirby, A. J.; Hollfelder, F. Org. Biomol. Chem. 2012, 10, 80958101.
(24) Duarte, F.; Bauer, P.; Barrozo, A.; Amrein, B. A.; Purg, M.; qvist, J.; Kamerlin, S. C. L. J.
Phys. Chem. B. 2014, 118, 4351-4362.
(25) Jonas, S., PhD Thesis, University of Cambridge, 2009.
(26) Florin, J.; Warshel, A. J. Am. Chem. Soc. 1997, 119, 5473-5474.

S10

S3. Figures & Tables

Figure S1: A schematic model of the octahedral dummy model used to describe the catalytic
metal center in the present work. In this model, the partial charge on the central atom is
redistributed such that it is delocalized equally onto each dummy particle, and the charge on the
central atom is corrected to maintain an overall charge of +2. For technical details of the model
and its validation, as well as the parameters used in the current work please see ref. 24.

S11

Figure S2: Time-averages of the root mean square deviation (in ) of the protein backbone
atoms (C, C, and N atoms) for the monomeric unit of the WT and manually generated mutant
forms of RlPMH with the phenyl p-nitrophenyl phosphonate substrate bound in the active site. In
this case only the monomeric unit is considered, as most of the atoms that belong the other units
are restrained to their crystallographic positions as outlined in the Methodology section.

S12

Figure S3: Time-averages of the root mean square deviation (in ) of the protein backbone
atoms (C, C, and N atoms) for the monomeric unit of the WT and manually generated mutant
forms of RlPMH with the ethyl p-nitrophenyl phosphate substrate bound in the active site. In this
case only the monomeric unit as considered, as most of the atoms that belong the other units are
restrained to their crystallographic positions as outlined in the Methodology section.
1

0.8

0.8

0.8

0.7

0.7

0.7

0.6
0.5

RMSD [A
]

0.9 PETN78A

RMSD [A
]

0.9 PETQ13A

0.6
0.5

0.5

0.4

0.4

0.3

0.3

0.3

2
3
time (ns)

0.2
0
5
1

2
3
time (ns)

0.2
5 0
1

0.9 PET107A

0.9 PETH218A

0.8

0.8

0.8

0.7

0.7

0.7

0.6
0.5

RMSD [A
]

0.9 PETY105A

0.6
0.5

0.4

0.3

0.3

0.3

2
3
time (ns)

0.2
5 0

2
3
time (ns)

2
3
time (ns)

0.9 K337A
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0

S13

2
3
time (ns)

0.5

0.4

2
3
time (ns)

0.6

0.4

0.2

RMSD [A
]

0.6

0.4

0.2

RMSD [A
]

0.9 PETWT

RMSD [A
]

RMSD [A
]

0.2
5
0

Figure S4: pH-rate profiles of kcat/KM for all six substrates considered in ref. 18: (A) pnitrophenyl phosphate (PNP) (B) ethyl p-nitrophenyl phosphate (PET), (C) diethyl p-nitrophenyl
phosphate (paraoxon), (D) phenyl p-nitrophenyl phosphonate (PPP), (E) p-nitrophenyl sulfate
monoester (PNS), and (F) phenyl p-nitrophenyl sulfonate monoester (PPS). Note as discussed in
the main text that the unusual pH rate profile for the phosphate triester, combined with the very
poor activity, makes us question whether this is a true promiscuous activity in the same active site
as for other substrates, and we have therefore not considered substrate (C) in the present work.
Reproduced with permission from Proceedings of the National Academy of Sciences U. S. A.18.

S14

Figure S5: Average P(S)-O and H-N/Olg distances along the reaction coordinate for the
hydrolysis of phenyl p-nitrophenyl phosphonate (PPP), ethyl p-nitrophenyl phosphate (PET), pnitrophenyl sulfate (PNS), phenyl p-nitrophenyl sulfonate (PPS), and the p-nitrophenyl
phosphate monoanion (PNPH) in the wild-type form of BcPMH. The position of the transition
state is marked with dashed lines. Color-coding: P-Olg (blue), P-Onuc (light-blue), N-H (green),
and H-Olg (light green). All distances are given in ngstrms (). As can be seen from this plot,
this is an asynchronous but concerted process, with initial nucleophilic attack on the P/S center,
followed by proton transfer to the leaving group oxygen.

S15

Figure S6: Average P(S)-O and H-N/Olg distances along the reaction coordinate for the
hydrolysis of phenyl p-nitrophenyl phosphonate (PPP) and ethyl p-nitrophenyl phosphate (PET)
in the K337 form of RlPMH. The position of the transition state is marked with dashed lines.
Color-coding: P-Olg (blue), P-Onuc (light-blue), N-H (green), and H-Olg (light green). All
distances are given in ngstrms ().

S16

Table S1: Kinetic parameters available for the PMHs being considered in this work. All values
were extracted from refs. 18,19,25. All measurements were made at pH 7.5 and 30 C. For the
reactions in solution, values for PPP were extracted from refs. 17,18, PET from ref. 12, PNS
from ref. 11, and PNPH from ref. 21. See discussion in Section S1.2 for estimates of the reaction
rates for the alkaline hydrolysis of PPP and PNPH. All the values for reactions not measured at
30 C were corrected in order to allow comparison.
RlPMH(e)

Solution
Substrate

kH2O (s-1)

kOH- (s-1)(d)

kcat (s-1)

kcat/ KM (M-1s-1)

kcatkH2O

kcatkOH-

PPP(a)

1.7 1011

7.5 10-8

16.0 1.0

(5.3 0.6) 103

9.4 1011

2.1 108

PET(b)

2.6 1013

8.0 10-8

9.5 0.6

(4.1 1.0) 103

3.7 1013

1.2 108

PNS

1.1 109

2.2 10-9

n.d.

3.5

n.d.

n.d.

PPS

-3

n.d.

9.9

n.d.

n.d.

0.11 0.01

(2.5 0.2) 102

2.3 107

5.8 104

5.5 10

PNPH(c)

4.3 10-9

3.4 10

1.9 10-6

Solution
kH2O (s )

kOH (s )

kcat (s )

kcat/ KM (M-1s-1)

kcatkH2O

kcatkOH-

PPP(a)

1.7 1011

7.5 10-8

2.73 0.06

(1.5 0.1) x 104

1.6 1011

3.6 107

PET(b)

2.6 1013

8.0 10-8

5.8 0.1

(9.2 0.6) x 103

2.2 1013

7.3 107

PNS

1.1 109

2.2 10-9

(4.0 0.1) 102

0.59 0.04

3.6 107

1.8 107

PPS

5.5 109

3.4 10-3

(1.2 0.1) 102

49 7

2.2 106

3.5

-9

-6

22 1

PNPH
(a)

BcPMH

Substrate

(c)

-1

4.3 10

-1

1.9 10

-1

(7.7 0.1) x 10

1.8 10

4.1 103

kOH- derived using kOH- for methyl p-nitrophenyl phosphonate from Behrman and coworkers17. The effect of

substitution of the methyl for phenyl group in the phosphonate was estimated by comparing available kH2O values for
both methyl and phenyl phosphonate17,18 (see Section S1.2 for details).
(b)

Derived from Purcell and Hengge12, by using the published temperature dependence for kOH- and extrapolation to

kH2O at pH 7.5([OH]=10-(14-7.5) =3.2 10-7 M).

(c)

kOH- for PNPH is immeasurable, since the phosphate will not be protonated under alkaline conditions (see

discussion in refs. 21,26). Estimation of this value was made based on methylated analogue (see ref. 21).
(d)

Values corrected for nucleophile concentration.

(e)

n.d. refers to values that have not been experimentally determined.

S17

Table S2: Kinetic parameters available for the RlPMH wild-type and several mutants. All values
were extracted from ref. 19. All measurements were made at pH 7.5 and 30 C.
PPP

PET

Variant

kcat (s-1)

kcat/ KM (M-1s-1)

kcat (s-1)

kcat/KM (M-1s-1)

Wild type

16.0 1.0

(5.3 0.6) x 103

9.5 0.6

(4.1 1.0) x 103

Q13A

26.0 3.0

(6.0 1.5) x 102

18.0 3.0

(3.6 1.3) x 102

N78A

1.5 0.2

(3.7 1.0) x 102

1.1 0.1

25.0 7.0

Y105A

2.7 0.2

99.0 12.0

> 0.17

4.6 1.9

T107A

2.4 0.1

(2.5 0.2) x 102

0.57 0.01

(1.2 0.1) x 102

H218A

1.0 0.1

0.11 0.01

0.30 0.02

5.2 0.3

K337A

12.0 1.0

(8.5 1.2) x 102

2.9 0.1

40.0 2.0

S18

Table S3: Partial charges (Q) of the nucleophile oxygen (Onuc), the central P/S atom plus all
atoms connected to it (three non-bridging oxygens for PET, PNS and PNPH and two nonbridging oxygens and the carbon atom of the spectator group for PPP and PPS) and the leaving
group oxygen (Olg) for the hydrolysis of all substrates studied in this work. The transition state
charges were calculated from a linear combination of the charges of the reactant and product
states for each substrate, which are included in the EVB parameters provided as Supporting
Information. A representation of the partition used is shown below (Rlg=pNO2Ph,
Rnuc=CH3CHOH and R=Ph (PPP/PPS), CH3 (PET) or H (PNPH)).

kcatKM

Onuc

Substrate

P(S)+Onb+OR(CR)

Olg

(M1 s1)(a)

RS

TS

QTS-RS

RS

TS

QTS-RS

RS

TS

QTS-RS

PPP

1.5 104

-1.0036

-0.7308

0.2728

-0.5081

-0.5549

-0.0468

-0.6099

-0.5704

0.0395

PET

9.2 10

-1.0036

-0.7286

0.2751

-0.7974

-0.8343

-0.0369

-0.5494

-0.5402

0.0092

PPS

49

-1.0036

-0.7322

0.2715

-0.0188

-0.0518

-0.0330

-0.4238

-0.4774

-0.0536

PNPH

22

-1.0036

-0.7396

0.2641

-0.9168

-0.9875

-0.0707

-0.6727

-0.6019

0.0708

PNS

0.59

-1.0036

-0.7534

0.2503

-0.5992

-0.6834

-0.0842

-0.5157

-0.5234

-0.0077

(a)

Taken from ref. 18.

S19

Table S4: Average P(S)-O and H-N/Olg distances at the transition state for the relevant group
transfer reaction in water and in the wild-type form of RlPMH and BcPMH. All distances are
given in ngstrms ().
Substrate
PPP
PET
PNS
PPS
PNPH

P(S)-Onuc
1.89 0.08
2.09 0.29
2.13 0.17
1.85 0.07
1.99 0.25

Water
P(S)-Olg
H-N(K337)
2.14 0.12 1.09 0.07
2.04 0.12 1.07 0.06
2.06 0.13 1.06 0.05
2.04 0.09 1.04 0.05
2.14 0.18 1.13 0.07

H-Olg
1.40 0.12
1.43 0.12
1.44 0.12
1.49 0.10
1.32 0.10

P(S)-Onuc
2.05 0.10
2.06 0.11
2.11 0.13
1.88 0.07
2.03 0.11

RlPMH
P(S)-Olg
H-N(K337)
1.97 0.08 1.08 0.07
1.94 0.08 1.10 0.07
2.08 0.12 1.08 0.07
1.99 0.08 1.26 0.09
2.00 0.11 1.12 0.08

H-Olg
1.40 0.10
1.39 0.08
1.43 0.10
1.19 0.09
1.37 0.11

P(S)-Onuc
2.07 0.11
2.09 0.13
2.12 0.13
1.84 0.06
2.04 0.11

BcPMH
P(S)-Olg
H-N(K337)
1.95 0.08 1.08 0.06
1.97 0.10 1.09 0.07
2.08 0.12 1.08 0.06
1.99 0.08 1.05 0.06
2.00 0.10 1.12 0.10

H-Olg
1.43 0.09
1.41 0.10
1.43 0.09
1.43 0.09
1.37 0.08

Substrate
PPP
PET
PNS
PPS
PNPH
Substrate
PPP
PET
PNS
PPS
PNPH

S20

Table S5: Average P-O distances, in , at the transition state for phenyl p-nitrophenyl
phosphonate (PPP) and ethyl p-nitrophenyl phosphate (PET). Comparison between the
background reactions modeled in a water droplet, the wild-type enzyme, and various active site
mutant forms of RlPMH.
PPP
Water
WT
Q13A
N78A
Y105A
T107A
H218A
WT(BcPMH)
K337A(a)
(a)

PET

P(S)-Onuc

P(S)-Olg

P(S)-Onuc

P(S)-Olg

1.89 0.08
2.09 0.11
2.07 0.11
2.06 0.11
2.02 0.09
2.03 0.10
2.03 0.10
2.07 0.11
1.99 0.09

2.14 0.12
1.96 0.09
1.94 0.09
2.02 0.09
1.99 0.08
1.97 0.08
1.98 0.08
1.95 0.08
1.97 0.08

2.09 0.29
2.06 0.11
2.11 0.14
2.12 0.13
2.12 0.14
2.03 0.10
2.07 0.12
2.09 0.13
2.02 0.12

2.04 0.12
1.94 0.08
1.96 0.10
1.95 0.09
1.98 0.10
1.98 0.09
1.99 0.10
1.97 0.10
1.98 0.09

For K337A variant, in the absence of K337 as a proton donor, we instead modeled

general-acid catalysis from H218, assuming this residue could become protonated once the
positive charge of the K337 is removed from its close proximity. For this model, the key
transition state distances in the background reaction are 2.02 0.12 (P-Onuc) and 1.98 0.09
(P-Olg). All reported values are averages and standard deviations over 10 individual
trajectories, as described in the Methodology section of the main text.

S21

Table S6: Average P(S)-O and H-N/Olg distances, in , at the transition state for concerted
mechanism of group and proton transfers in water, wild-type and K337A mutant forms of
RlPMH. Since H218 plausibly takes the role of K337 and acts as a general acid for the K337A
variant, two possible mechanisms involving distinct acids were modeled.
SubstrateGeneral acid
PPP-LYS
PPP-HIS
PET-LYS
PET-HIS

Water
P-Onuc
1.89 0.08
2.02 0.12
2.09 0.29
1.95 0.17

P-Olg
2.14 0.12
1.98 0.09
2.04 0.12
2.08 0.13

SubstrateSystem
PPP-WT
PPP-K337A
PET-WT
PET-K337A

H-Nacid
1.09 0.07
1.23 0.09
1.07 0.06
1.29 0.07

H-Olg
1.40 0.12
1.21 0.10
1.43 0.12
1.17 0.12

RlPMH
P-Onuc
2.05 0.10
1.99 0.09
2.06 0.11
2.04 0.13

P-Olg
1.97 0.08
1.97 0.08
1.94 0.08
1.98 0.11

S22

H-Nacid
1.08 0.07
1.26 0.09
1.10 0.07
1.24 0.08

H-Olg
1.40 0.10
1.19 0.09
1.39 0.08
1,22 0.10

Table S7: An overview of the correlation between selected physical properties(a) and the number
of known catalytic activities for several members of the AP superfamily. The enzymes chosen
for analysis were Escherichia coli alkaline phosphatase (AP, PDB code 1ALK), Burkholderia.
caryophilli phosphonate monoester hydrolase (PMH, PDB code 2W8S), Xanthomonas.
axonopodis

nucleotide

pyrophosphatase/phosphodiesterase

(NPP,

PDB

code

2GSN),

Pseudomonas aeruginosa arylsulfatase (PAS, PDB code 1HDH), Homo sapiens lysosomal
arylsulfatase A (ASA, PDB code 1AUK), Homo sapiens lysosomal arylsulfatase B (ASB, PDB
code 1FSU) and Bacillus stearothermophilus cofactor-independent phosphoglycerate mutase
(iPGM, PDB code 1EJJ).
AP

PMH

NPP

PAS

ASA

ASB

iPGM

1870.7

1215

1016.9

756.7

539.8

431.9

151.2

Polar SASA (2)

320.0

240.4

238.5

87.2

181.3

106.6

39.2

Apolar SASA ( )

239.2

376.9

289.0

416.2

141.4

116.9

20.3

Total SASA (2)

559.2

617.3

527.5

503.4

322.7

223.5

59.6

Activities

Volume (3)

Physical properties were obtained from the Voronoi tessellation analysis implemented in the Fpocket 2 software

package26. The parameters used in this analysis are presented in Table S8. The parameters not mentioned herein
were set to the Fpocket 2 default values. 1,000,000 points were used for the volume and surface sampling.

Table S8: Parameters used in Fpocket 226 to perform the analysis presented in Table S7 and
Figure 9 of the main text. All parameters not mentioned here were set to their default values.
AP

PMH NPP PAS ASA ASB iPGM

Number of the pocket analyzed

Smallest sphere radius ()

3.0

3.5

3.0

2.75

3.0

3.0

3.0

Largest sphere radius ()

7.0

7.0

7.0

6.75

7.0

7.0

7.0

Minimum number of spheres per pocket 35.0

15.0

15.0

35.0

35.0

35.0

35.0

S23

S4. Empirical Valence Bond Parameters Used in this Work

The EVB parameters used in this work were obtained as outlined in the Methodology section
in the main text. All parameters not listed here are standard OPLS-AA parameters for the
relevant atom types. For details of the EVB approach we refer the reader to refs. 1-3. The offdiagonal elements, which describe the coupling between the two EVB diabatic states, can be
represented by simple-exponential functions of the form:
H!" = A!" exp [ (r!" r! )]

(1)

where rij denotes the distance between atoms i and j and r0 denotes the equilibrium distance or, as
in this work, by a constant, where the parameter is set equal to zero. The gas-phase shift, i, is
a constant that can be added to either parabola in the reference state in order to reproduce the
overall free energy of the background reaction. In the present work, i was always added to State
II (for definitions of the states see the schema below). The off-diagonal elements (Hij) and gasphase shift (i) values used in this work to calibrate the reference reactions are presented below.
The same parameters were then used unchanged in all enzyme runs.

Table S9: EVB mapping parameters used in this worka.

Substrate
PPP
PET
PNS
PPS
PNPH
PPP (K337A)
PET (K337A)
a

Hij
91.05
120.90
134.00
103.30
99.00
89.40
123.00

i
-207.30
-199.90
-179.50
-179.10
-201.00
-163.25
-155.70

Both Hij and i are constants. All values are given in kcal mol-1.

S24

The different valence bond states used to model the PMH reactions in this work are illustrated
below, and the R groups on each substrate studied correspond to those shown in Figure 2 of the
main text.

State II

PPP/PPS*

State I

PET

*X=P or S

S25

State II

PNPH

PNS

State I

S26

Overview of parameters
Van der Waals parameters used for atoms constituting the reacting part of the system
Type

Ai

Bi

(kcal1/2mol1/2
6)

(kcal1/2mol1/2
3)

Ci

(kcal mol-1)

(2)

A1-4

B1-4

(kcal1/2mol1/2
3)

(kcal1/2mol1/2
3)

mass
(a.u.)

C2
1802.24
34.18
91.0
2.5
1274.38
24.17
12.01
CT
944.52
22.03
91.0
2.5
667.88
15.58
12.01
CTB
724.36
18.76
91.0
2.5
667.88
15.58
12.01
C(A/R/V/W/X)
1059.13
23.67
91.0
2.5
748.92
16.74
12.01
H(3/O)
0.00
0.00
5.0
2.5
0.00
0.00
1.01
HA
69.58
4.91
5.0
2.5
49.20
3.47
1.01
HC
49.20
3.47
5.0
2.5
34.79
2.45
1.01
HCDDP
109.18
6.99
5.0
2.5
77.20
4.94
1.01
N(A/B/3)
971.75
28.31
60.0
2.5
687.13
20.02
14.01
PNZ
816.43
23.78
60.0
2.5
577.31
16.82
14.01
NT
1064.97
29.63
60.0
2.5
753.05
20.96
14.01
OP
976.93
31.26
90.0
2.5
690.79
22.10
16.00
OTY
690.37
23.86
53.0
2.5
488.17
16.87
16.00
OH
760.65
25.05
53.0
2.5
537.86
17.71
16.00
OP1
445.13
18.25
90.0
2.5
314.75
12.91
16.00
OP2
873.79
27.96
53.0
2.5
617.86
19.77
16.00
O2
616.44
23.77
90.0
2.5
435.89
16.81
16.00
OHB
703.98
24.09
53.0
2.5
497.79
17.71
16.00
PON
554.63
21.39
53.0
2.5
392.12
15.12
16.00
P
2447.79
46.79
43.0
2.5
1730.85
33.09
30.97
S
2001.57
44.74
35.0
2.5
1415.32
31.64
32.06
Soft pair repulsion energy between atoms i and j: Vsoft = Ci Cj exp (-ijrij); rij denotes the distance ()
between atom i and j. This potential replaces the vdW interactions between atoms forming and
breaking bonds.

S27

#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

Atom Types in Different VB Statesa

PPP/PPS
PET
PNS
State I
State II
State I
State II
State I
State II
CTB
CTB
CTB
CTB
CTB
CTB
HCDDP
HC
HCDDP
HC
HCDDP
HC
OP
OP1
OP
OP1
OP
OP1
OHB
OHB
OHB
OHB
OHB
OHB
HO
HO
HO
HO
HO
HO
P/S
P/S
P
P
S
S
OP1
OTY
OP1
OTY
OP1
OTY
OP2
OP2
OP2
OP2
OP2
OP2
OP2
OP2
OP2
OP2
OP2
OP2
CA
CA
OP1
OP1
OP2
OP2
CA
CA
CA
CA
CA
CA
HA
HA
CA
CA
CA
CA
CA
CA
HA
HA
HA
HA
HA
HA
CA
CA
CA
CA
CA
CA
HA
HA
HA
HA
PNZ
PNZ
CA
CA
CA
CA
PON
PON
PNZ
PNZ
PNZ
PNZ
PON
PON
PON
PON
PON
PON
CA
CA
PON
PON
PON
PON
HA
HA
CA
CA
CA
CA
CA
CA
HA
HA
HA
HA
HA
HA
CA
CA
CA
CA
CA
CA
HA
HA
HA
HA
CA
CA
CT
CT
MN6
MN6
HA
HA
HC
HC
D61
D61
CA
CA
HC
HC
D62
D62
HA
HA
CT
CT
D63
D63
CA
CA
HC
HC
D61
D61
HA
HA
HC
HC
D62
D62
CA
CA
HC
HC
D63
D63
HA
HA
MN6
MN6
CT
CT
CA
CA
D61
D61
HC
HC
HA
HA
D62
D62
HC
HC
MN6
MN6
D63
D63
C2
C2
D61
D61
D61
D61
O2
O2
D62
D62
D62
D62
O2
O2
D63
D63
D63
D63
CT
CT
D61
D61
CT
CT
HC
HC
D62
D62
HC
HC
HC
HC
D63
D63
HC
HC
N3
NT

S28

PNPH
State I
State II
CTB
CTB
HCDDP
HC
OP
OP1
OHB
OHB
HO
HO
P
P
OP1
OTY
OP2
OP2
OP2
OP2
OP1
OP1
CA
CA
CA
CA
HA
HA
CA
CA
HA
HA
CA
CA
PNZ
PNZ
PON
PON
PON
PON
CA
CA
HA
HA
CA
CA
HA
HA
HO
HO
MN6
MN6
D61
D61
D62
D62
D63
D63
D61
D61
D62
D62
D63
D63
CT
CT
HC
HC
HC
HC
C2
C2
O2
O2
O2
O2
CT
CT
HC
HC
HC
HC

41
CT
CT
C2
C2
H3
42
HC
HC
O2
O2
H3
43
HC
HC
O2
O2
H3
44
C2
C2
CT
CT
45
O2
O2
HC
HC
46
O2
O2
HC
HC
47
CT
CT
N3
NT
48
HC
HC
H3
H
49
HC
HC
H3
H
50
N3
NT
H3
HO
51
H3
H
52
H3
H
53
H3
HO
a
The metal center was included only in the enzyme reaction.

S29

H
H
HO
-

N3
H3
H3
H3
-

NT
H
H
HO
-

Atom Types in Different VB States (K337A variant)

PPP
PET
Atom
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

State I

State II

State I

State II

CTB
HCDDP
OP
OHB
HO
P
OP1
OP2
OP2
CA
CA
HA
CA
HA
CA
PNZ
PON
PON
CA
HA
CA
HA
CA
CA
HA
CA
HA
CA
HA
CA
HA
CA
HA
MN6
D61
D62
D63
D61
D62
D63

CTB
HC
OP1
OHB
HO
P
OTY
OP2
OP2
CA
CA
HA
CA
HA
CA
PNZ
PON
PON
CA
HA
CA
HA
CA
CA
HA
CA
HA
CA
HA
CA
HA
CA
HA
MN6
D61
D62
D63
D61
D62
D63

CTB
HCDDP
OP
OHB
HO
P
OP1
OP2
OP2
OP1
CA
CA
HA
CA
HA
CA
PNZ
PON
PON
CA
HA
CA
HA
CT
HC
HC
CT
HC
HC
HC
MN6
D61
D62
D63
D61
D62
D63
CT
HC
HC

CTB
HC
OP1
OHB
HO
P
OTY
OP2
OP2
OP1
CA
CA
HA
CA
HA
CA
PNZ
PON
PON
CA
HA
CA
HA
CT
HC
HC
CT
HC
HC
HC
MN6
D61
D62
D63
D61
D62
D63
CT
HC
HC

S30

41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58

CT
HC
HC
C2
O2
O2
CT
HC
HC
CX
NA
H
CR
HA
NA
H
CX
HA

CT
HC
HC
C2
O2
O2
CT
HC
HC
CW
NA
H
CR
HA
NB
HO
CV
HA

S31

C2
O2
O2
CT
HC
HC
CX
NA
H
CR
HA
NA
H
CX
HA
-

C2
O2
O2
CT
HC
HC
CW
NA
H
CR
HA
NB
HO
CV
HA
-

PPP
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44

State I
0.3723
-0.0295
-1.0036
-0.8635
0.5243
1.2216
-0.6099
-0.7747
-0.7747
0.7131
-0.3838
0.1920
-0.1292
0.1806
-0.1090
0.8566
-0.5217
-0.5217
-0.1292
0.1806
-0.3838
0.1920
-0.1803
-0.0793
0.1337
-0.1772
0.1166
-0.1155
0.1094
-0.1772
0.1166
-0.0793
0.1337
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000

State II
0.2108
0.0935
-0.4580
-0.6129
0.4148
1.0964
-0.5309
-0.7906
-0.7906
0.4265
-0.3070
0.1897
-0.1283
0.1883
-0.0141
0.7917
-0.4734
-0.4734
-0.1283
0.1883
-0.3070
0.1897
-0.1168
-0.0605
0.1306
-0.2058
0.1147
-0.1089
0.1042
-0.2058
0.1147
-0.0605
0.1306
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000

PET
State I
0.3723
-0.0295
-1.0036
-0.8635
0.5243
1.2137
-0.5494
-0.7468
-0.7468
-0.5175
0.6585
-0.3560
0.1780
-0.1194
0.1756
-0.1086
0.8405
-05174
-0.5174
-0.1194
0.1756
-0.3560
0.1780
0.3910
-0.0342
-0.0342
-0.1943
0.0355
0.0355
0.0355
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.1900

State II
0.2192
0.0734
-0.4535
-0.6134
0.4219
1.1989
-0.5309
-0.7901
-0.7901
-0.4899
0.4265
-0.3070
0.1897
-0.1283
0.1883
-0.0141
0.7917
-0.4734
-0.4734
-0.1283
0.1883
-0.3070
0.1897
0.4532
-0.0491
-0.0491
-0.2956
0.0546
0.0546
0.0546
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.0600

Atomic Charges
PNS
State I
0.3723
-0.0295
-1.0036
-0.8635
0.5243
1.3343
-0.5157
-0.6445
-0.6445
-0.6445
0.5849
-0.3161
0.1753
-0.1289
0.1776
-0.0991
0.8435
-0.5151
-0.5151
-0.1289
0.1776
-0.3161
0.1753
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.1900
0.0600
0.0600
-0.3000
0.3300
0.3300
0.3300
-

S32

State II
0.6438
-0.0503
-0.5031
-0.7302
0.4073
1.2610
-0.5311
-0.6762
-0.6762
-0.6762
0.4265
-0.3070
0.1897
-0.1283
0.1883
-0.0141
0.7917
-0.4734
-0.4734
-0.1283
0.1883
-0.3070
0.1897
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.0600
0.0600
0.0600
-0.9000
0.3600
0.3600
0.3882
-

PNPH
State I
0.3723
-0.0295
-1.0036
-0.8635
0.5243
1.3684
-0.6727
-0.7876
-0.7876
-0.7100
0.8031
0.4430
0.2161
-0.0978
0.1741
-0.1265
0.8550
-0.5222
-0.5222
-0.0978
0.1742
-0.4430
0.2161
0.4034
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.1900
0.0600
0.0600
-0.3000
0.3300
0.3300
0.3300

State II
0.2460
0.1032
-0.4755
-0.6535
0.4261
1.3026
-0.5311
-0.8168
-0.8168
-0.7272
0.4265
-0.3070
0.1897
-0.1283
0.1883
-0.0141
0.7917
-0.4734
-0.4734
-0.1283
0.1883
-0.3070
0.1897
0.4119
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000
-0.8000
-0.8000
0.0600
0.0600
0.0600
-0.9000
0.3600
0.3600
0.3882

PPS
State I
0.3723
-0.0295
-1.0036
-0.8635
0.5243
1.1656
-0.4238
-0.5318
-0.5318
0.3940
-0.2235
0.1897
-0.1879
0.2014
0.0291
0.8003
-0.4664
-0.4664
-0.1879
0.2014
-0.2235
0.1897
-0.1208
-0.0923
0.1533
-0.1406
0.1509
-0.0976
0.1476
-0.1406
0.1509
-0.0923
0.1533
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000

State II
0.6318
-0.0071
-0.4607
-0.6585
0.4146
0.9738
-0.5309
-0.5270
-0.5270
0.4265
-0.3070
0.1897
-0.1283
0.1883
-0.0141
0.7917
-0.4734
-0.4734
-0.1283
0.1883
-0.3070
0.1897
-0.0045
-0.1008
0.1637
-0.1614
0.1505
-0.0811
0.1417
-0.1614
0.1505
-0.1008
0.1637
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
0.0600
0.0600
0.7000

45
46
47
48
49
50
51
52
53

-0.8000
-0.8000
0.1900
0.0600
0.0600
-0.3000
0.3300
0.3300
0.3300

-0.8000
-0.8000
0.0600
0.0600
0.0600
-0.9000
0.3600
0.3600
0.3882

0.0600
0.0600
-0.3000
0.3300
0.3300
0.3300
-

0.0600
0.0600
-0.9000
0.3600
0.3600
0.3882
-

S33

-0.8000
-0.8000
0.1900
0.0600
0.0600
-0.3000
0.3300
0.3300
0.3300

-0.8000
-0.8000
0.0600
0.0600
0.0600
-0.9000
0.3600
0.3600
0.3882

#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41

Atomic Charges (K337A mutant mechanism)

PPP
PET
State I
State II
State I
State II
0.3723
0.2108
0.3723
0.2192
-0.0295
0.0935
-0.0295
0.0734
-1.0036
-0.4580
-1.0036
-0.4535
-0.8635
-0.6129
-0.8635
-0.6134
0.5243
0.4148
0.5243
0.4219
1.2216
1.0964
1.2137
1.1989
-0.6099
-0.5309
-0.5494
-0.5309
-0.7747
-0.7906
-0.7468
-0.7901
-0.7747
-0.7906
-0.7468
-0.7901
0.7131
0.4265
-0.5175
-0.4899
-0.3838
-0.3070
0.6585
0.4265
0.1920
0.1897
-0.3560
-0.3070
-0.1292
-0.1283
0.1780
0.1897
0.1806
0.1883
-0.1194
-0.1283
-0.1090
-0.0141
0.1756
0.1883
0.8566
0.7917
-0.1086
-0.0141
-0.5217
-0.4734
0.8405
0.7917
-0.5217
-0.4734
-05174
-0.4734
-0.1292
-0.1283
-0.5174
-0.4734
0.1806
0.1883
-0.1194
-0.1283
-0.3838
-0.3070
0.1756
0.1883
0.1920
0.1897
-0.3560
-0.3070
-0.1803
-0.1168
0.1780
0.1897
-0.0793
-0.0605
0.3910
0.4532
0.1337
0.1306
-0.0342
-0.0491
-0.1772
-0.2058
-0.0342
-0.0491
0.1166
0.1147
-0.1943
-0.2956
-0.1155
-0.1089
0.0355
0.0546
0.1094
0.1042
0.0355
0.0546
-0.1772
-0.2058
0.0355
0.0546
0.1166
0.1147
-1.0000
-1.0000
-0.0793
-0.0605
0.5000
0.5000
0.1337
0.1306
0.5000
0.5000
-1.0000
-1.0000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
0.5000
-0.2200
-0.2200
0.5000
0.5000
0.0600
0.0600
0.5000
0.5000
0.0600
0.0600
-0.2200
-0.2200
0.7000
0.7000

S34

42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58

0.0600
0.0600
0.7000
-0.8000
-0.8000
-0.0050
0.0600
0.0600
0.2150
-0.5400
0.4600
0.3850
0.1150
-0.5400
0.4600
0.2150
0.1150

0.0600
0.0600
0.7000
-0.8000
-0.8000
-0.0050
0.0600
0.0600
0.0150
-0.5700
0.4200
0.2950
0.1150
-0.4900
0.3882
-0.0150
0.1150

S35

-0.8000
-0.8000
-0.0050
0.0600
0.0600
0.2150
-0.5400
0.4600
0.3850
0.1150
-0.5400
0.4600
0.2150
0.1150

-0.8000
-0.8000
-0.0050
0.0600
0.0600
0.0150
-0.5700
0.4200
0.2950
0.1150
-0.4900
0.3882
-0.0150
0.1150

Bond Parameters for Covalent Bonds of the Reacting Part of the System
De

r0
kb
b
Bond Type
(kcalmol-1)

(-2)

()

(kcalmol-1-2)

()

95.0
105.0
110.0
110.0
-

2.0
2.0
2.0
2.0
-

1.61
1.599
1.01
0.94
-

734.0
764.0
954.0
976.0
854.0
820.0

1.471
1.448
1.343
1.335
1.381
1.394

P-OP
S-OP
N3-H3 (Morse)
OTY-HO
N3-H3
NT-H
CR-NA
CR-NB
CX-NA
CV-NB

Morse bonds: VMorse =De {1-exp[- (rij-r0)]}2; Harmonic bond: VHarmonic =0.5kb (rij-b)2

Angle Parameters of the Reacting Part of the System

Angle Type

ka (kcalmol-1rad-2)

0 ()

P(S)-OP-CA / P-OP-CT
OP1-P(S)-OP2
OP1-P(S)-CA
CA-OTY-HO
H3-N3-H3
H-NT-H
CX-NA-CR
CR-NB-CV
CX-CX-NA
CW-CV-NB
HA-CX-CX
HA-CV-CW
CX-CT-CT
CT-CW-CV
CR(CX)-NA-H
O2-O2-MN6

200.0
200.0
90.0
70.0
70.0
87.2
140.0
140.0
140.0
140.0
70.0
70.0
126.0
140.0
70.0
50.0

120.50
108.23
109.50
113.00
109.50
106.40
109.80
110.00
106.30
111.00
130.70
128.20
114.00
130.70
120.00
180.00

Harmonic angle potential: Va = ka(-0)2

S36

Torsion Parameters of the Reacting Atoms of the System

Torsion
V1
V2
V3
Type
0.5barrier height (kcalmol-1)
P(S)-OP1-CA-CA / P-OP1-CT-CT
0.0000
1.4950
0.0000
OP1(2)-P(S)-OP1-CA / OP2-P(S)-OP1-CT
0.0000
0.0000
0.2810
P(S)-OP1-CT-HC
0.0000
0.0000
0.1780
OH-CT-OP1-P
0.0000
0.0000
0.0000
OP1-CT-CT-HC
0.0000
0.0000
0.2340
HO-OHB-CTB-OP1
-0.1780
-0.0870
0.2460
HO-OHB-CTB-HC(HCDDP)
0.0000
0.0000
0.2250
HO-OHB-CTB-OP(OP1)
-0.6280
-0.9030
0.0020
NA-CR-NA-H(CX) / HA-CR-NA-CX
0.0000
2.3250
0.0000
CX-CX-NA-CR(H)
0.0000
1.4000
0.0000
HA-CX-NA-H(CR)
0.0000
1.6000
0.0000
CV-NB-CR-NA(HA)
0.0000
5.0000
0.0000
CX(HA)-NA-CR-NA
0.0000
2.3250
0.0000
CX-NA-CR-NA(HA)
0.0000
2.4000
0.0000
CR-NA-CX-HA
0.0000
1.6000
0.0000
N3-CT-CT-CT
1.3660
-0.1150
0.2430
NT-CT-CT-CT
1.1960
-0.3370
0.2750
CT-CT-N3-H3
0.0000
0.0000
0.1740
CT-CT-NT-H
-0.0900
-0.2090
-0.2090
HC-CT-N3-H3
0.0000
0.0000
0.1310
HC-CT-NT-H
0.0000
0.0000
0.2000
HC-CT-CT-N3
0.0000
0.0000
0.1920
HC-CT-CT-NT
-0.5070
-0.3550
0.2370

Torsion force: Va = V1(1+cos)/2 + V2(1-cos2)/2 + V3(1+cos3)/2

S37