Dephosphorylation reaction

In the dephosphorylation reaction (of which the geometry parameters are

presented in Table 3.12 and Figure 3.25) with the diethyl-substituted

phosphor-adduct (from reaction with dECP), the landscape was more

complex. Three different reactant conformers, three TS1 conformers and

four intermediate conformers were identified – and in all probability

many more exist. The multiple conformers (see Figure 3.24) were found

during an attempt to make the TS1 geometry more similar to that of TS2

with the aim of obtaining a single intermediate that linked TS1 and TS2

into a single reaction path. The TS1 initially optimised was labelled TS1

1

; a

modification of its geometry to place His471 in a similar position to TS2

was labelled TS1

2,

and a subsequent TS search in which the active site

structure obtained for TS2 was used – modifying the position of the

reacting atoms and then optimising it with only Cα frozen – yielded TS1

3

.

Each intermediate and reactant geometry were located from an IRC search

from the respective TS1 geometry. As was expected, Intermediate(1)

1

– the

number between parenthesis indicates whether it was optimised from TS1

or TS2 – was the most different in terms of geometry from Intermediate(2),

and Intermediate(1)

3

was the most similar, since the active site

conformation of TS2 was used to identify TS1

3

. The RMSD between

conformers ranges between 0.4 and 1.0 Å (Table 3.13). However, the

intermediate conformers more similar to Intermediate(2) resulted in

higher energy barriers than this conformer, rather than more similar ones

(see Table 3.14). Clearly, obtaining a complete reaction path is more

difficult than simply adjusting the position of one residue. The modified

TS1 structures and their corresponding reactant geometries did not lead to

lower energy barriers for the first step, which suggests that what really

happens is a rearrangement of the intermediate, from the structure

obtained from TS1, into the structure that precedes TS2. Unfortunately,

time constraints did not make it possible to search for a transition between

these two intermediates as part of this project. The larger system,

however, showed that the overall barrier for dephosphorylation was

overestimated by the small system, as is discussed next (also see Figure

3.26 for a comparison of geometries).

Reactant

TS1

Intermediate

TS2

Product

P - O

wat

3.8

2.0

1.8

1.7

1.6

P - Oγ

1.6

1.7

1.7

2.0

3.8

H

wat

- OD

1.8

1.2

1.0

1.0

1.0

H

wat

- O

wat

1.0

1.2

1.5

1.6

2.6

Hγ - Oγ

3.9

3.7

2.1

1.3

1.0

Hγ – Nε

1.0

1.0

1.0

1.3

2.6

Hδ - OD

1.0

1.1

1.1

1.5

2.3

Hδ - Nδ

1.6

1.6

1.6

1.1

1.1

O

P

- H

219

3.3

3.5

4.4

4.7

4.7

O

P

- H

136

4.0

3.5

5.4

5.8

5.9

O

P

- H

137

2.6

2.7

4.2

4.6

5.3

Table 3.12. Principal geometry parameters of the dephosphorylation reaction

(dECP). The data show a concerted attack of the water molecule on the

phosphorus and proton transfer to Asp137 (TS1) and phosphorus-oxygen

bond break with concerted transfer of a proton from His471 to Ser218

(TS2). The geometries in this reaction path correspond to the species that

gave the lowest energy barriers. Distances in Å.

Conformer of intermediate

RMSD (all atoms)

Intermediate(1)

1

- Intermediate(2)

1

1.0

Intermediate(1)

2

- Intermediate(2)

1

0.7

Intermediate(1)

3

- Intermediate(2)

1

0.5

Intermediate(1)

1

- Intermediate(1)

2

1.0

Intermediate(1)

1

- Intermediate(1)

3

1.0

Intermediate(1)

2

- Intermediate(1)

3

0.4

Table 3.13. RMSD of intermediate conformers (dephosphorylation, large

system). The all-atoms RMSD between different conformers of the

intermediate shows significant variations ranging between 0.4 and 1.0 Å.

The subscripts have the same meaning as detailed above.

Figure 3.24.

Multiple structures of stationary points of the dephosphorylation

reaction.

A.

Reactant structures R

1

(green) and R

2

(blue).

B. Reactant

structures R

1

(green) and R

2

(magenta). C. Superimposition of R

1

, R

2

and

R

3

. D. TS1 structures TS1

1

(green) and TS1

2

(blue). E. TS1 structures TS1

1

(green) and TS1

3

(magenta). F. Superimposition of TS1

1

, TS1

2

and TS1

3

. G.

Intermediate structures I(2) (orange) and I(1)

1

(green). H. Intermediate

structures I(2) (orange) and I(1)

2

(blue). I. Intermediate structures I(2)

(orange) and I(1)

3

(magenta).

Species considered

Barrier

Large system

Small system

TS1

1

– Reactant

1

ΔE1

36

22

TS1

2

– Reactant

2

ΔE1

70

-

TS1

3

– Reactant

3

ΔE1

63

-

TS2 - Intermediate(1)

1

ΔE2

37

-

TS2 - Intermediate(2)

ΔE2

23

60

TS2 - Intermediate(1)

2

ΔE2

42

-

TS2 - Intermediate(1)

3

ΔE2

46

-

Table 3.14.

Energy barriers (dephosphorylation reaction of large system)

calculated using different geometries. The subscripts indicate the following:

1

Geometries optimised using the X-ray structure as a starting point.

2

Geometries obtained after placing His471 in the same position as in TS2.

3

Using the optimised geometry of TS2, the position of the reacting atoms

was modified and after a TS search a new TS1 conformer was obtained. (1)

and (2) have the same meaning as in Table 3.13 (above). Here too the

energy barriers calculated for the small system are included for

comparison.

Figure 3.25. Geometry coordinates of the dephosphorylation of the large system. A. Reactant. B. TS1. C. Intermediate. D. TS2. E. Product.

The geometries presented here, and in all other figures (unless otherwise specified) correspond to the conformers that gave the

lowest energy barriers. Distances in Å.

Figure 3.26. Comparison of structures of the dephosphorylation reaction for large and small systems. A. Reactant. B. TS1. C. Intermediate.

D. TS2. E. Product. Green = small system, magenta = large system.

The energy barriers calculated between the various reactants, transition

states and intermediates depend on the conformer chosen and range

between 36 and 70 kJ/mol (ΔE1) and between 23 and 46 kJ/mol (ΔE2) (see

Table 3.14 for energy comparison and Table 3.12 for geometry). The lowest

barriers calculated by this method are 36 kJ/mol for the first step and 23

kJ/mol for the second.

Evidence of the importance of the oxyanion hole in decreasing the energy

barrier of these reactions comes, rather unexpectedly, from the multiple

geometries of TS1 and reactant discussed earlier. A big difference between

the conformers is the distance between the phosphoryl oxygen and the

oxyanion hole (Table 3.15). The lowest ΔE1 energy barrier is that provided

by TS1

1

and the corresponding reactant, R

1

. The second lowest barrier is

that of TS1

3

with R

3

, and the highest barrier is that calculated with the

conformers TS1

2

and R

2

(Table 3.16). As is can be seen in Table 3.15, the

phosphoryl oxygen is in much closer contact with the oxyanion hole in

TS1

1

than in TS1

2

and TS1

3

. This would stabilise the transition state and

explain the lower energy barrier. Furthermore, although TS1

2

and TS1

3

have virtually the same parameters for these distances, in R

2

the

phosphoryl oxygen is much closer to one of the three members of the

oxyanion hole than in R

3

(while the other two are at the same distance). If

the reactant is stabilised by the oxyanion hole, the energy barrier for the

pair TS1

2

– R

2

would be higher than that of the pair TS1

3

– R

3

(although not

as large a difference as with respect to TS1

1

since in this case only one

distance is affected). This is indeed what is observed in the energy barriers

calculated. Therefore, although a complete reaction path on a single

coordinate could not be obtained, an attempt to solve the problems caused

by multiple possible pathways led to evidence that the oxyanion hole

plays a role in stabilising the system.

TS1

1

TS1

2

TS1

3

Reactant

1

Reactant

2

Reactant

3

2.0

2.0

2.0

P - O

wat

3.6

3.8

3.8

1.7

1.7

1.7

P - Oγ

1.6

1.6

1.6

1.2

1.2

1.2

H

wat

- OD

1.8

1.8

1.8

1.2

1.2

1.2

H

wat

- O

wat

1.0

1.0

1.0

3.7

2.3

1.9

Hγ - Oγ

3.9

2.5

2.9

1.0

1.0

1.0

Hγ – Nε

1.0

1.0

1.0

1.1

1.1

1.1

Hδ - OD

1.0

1.1

1.0

1.6

1.5

1.5

Hδ - Nδ

1.6

1.5

1.6

3.5

4.5

4.5

O

P

- H

219

3.3

4.4

4.4

3.5

5.4

5.4

O

P

- H

136

4.0

5.3

5.3

2.7

4.3

4.4

O

P

- H

137

2.6

2.6

4.3

Table 3.15. Relevant geometry (distance) parameters of the different conformers

of TS1 and reactant. Distance between different atoms in the multiple

conformers described in the text. Distances in Å.

Conformers

ΔE1 (kJ/mol)

TS11 - R1

36

TS12 - R2

70

TS13 -R3

54

Table 3.16.

Energy barriers obtained with the multiple conformers. The

subscript indicates the respective conformer (as detailed in text).

The geometries of the small and large system resemble each other more

closely in the dephosphorylation reaction than they did in

phosphorylation. Dephosphorylation was found to have very similar

distance coordinate parameters in the small and large systems (see Tables

3.9 and 3.16). A few exceptions are the distances between Hγ and Oγ in

TS1 and intermediate, those between Hγ and Nε in TS1 and product, and

those between H

wat

and O

wat

in the product. The barriers calculated for

each system, however, are highly different, which translates to very

different calculated reaction rates (summarised in Table 3.17). The small

systems led to a difference in predicted reaction rates of 272,000-fold

between phosphorylation and dephosphorylation (with phosphorylation

being the fastest), while the large system predicts that the difference in

rate between phosphorylation and dephosphorylation is a factor of 426.

This is in reasonable agreement with the experimental result (ca. 1600

fold) if it is considered that entropic differences are neglected in the

quantum cluster calculations. These results are encouraging, indicating

that the larger system is the appropriate size to reproduce experimental

results.

Small system Large system

Phosphorylation

5.12E+07

1.29E+09

Dephosphorylation

1.89E+02

3.04E+06

Table 3.17. Enzyme reaction rates obtained with different size of systems. Rates

in units of s

-1

.

Attempts to obtain a lower energy path by modifying the structures were

unsuccessful, however, this does not mean that alternative paths do not

exist. Many pathways are indeed possible, but structural constraints

imposed by the enzyme can limit pathway choice. One possibility for

future work is to explore different conformations (e.g. using umbrella

sampling methods) and the barriers they are associated to, with the aim of

finding a conformation, if possible, that makes the reaction faster and

finding out whether the enzyme can be forced into such conformation by

engineering.

In document Computational studies of the E3 carboxylesterase from Lucilia cuprina (Page 97-103)