Dephosphorylation system

Parathion and dichlorvos have dimethyl side chains, therefore the

phosphoadduct has dimethyl side chains, whereas dECP and diazinon

have diethyl side chains, and therefore the phosphoadduct has diethyl

side chains. For this reason, the dephosphorylation study would be made

of two systems instead of four. In order to have more data points for

comparison, two new dephosphorylation systems, the E3-VX(R) and E3-

VX(S) adducts, were introduced. For these, VX was docked to E3 (see

Chapter 4) and manually moved to a distance suitable for attack.

During examination of the converged structures of the dephosphorylation

system for the diethyl and dimethyl substrates it was noticed that in TS2

the His471 residue moves closer to the Ser218, in order to transfer a proton

to the serine oxygen atom. This contact still exists, as a hydrogen bond, in

the intermediate. There is no such hydrogen bond, however, in the X-ray

structure of the free enzyme. It is also not necessary in TS1, and it is

therefore also absent from the intermediate as determined by following

the IRC from TS1. Hence, two different intermediate geometries with

different energies are obtained from IRC of TS1 and TS2.

In the case of the diethyl substrate, the barriers initially obtained were 52

kJ/mol for the first step of the reaction and 60 kJ/mol for the second. It is

worth noting that, because of the occurrence of different intermediate

geometries as described in the previous paragraph, calculating ΔE2 using

the intermediate obtained from TS1 resulted in an erroneous negative

barrier, which means this intermediate geometry has higher energy than

the transition state. Because of this error, the position of His471 in the

initial structure used for TS1 search was changed to match its position in

leads to a geometry of the intermediate that is virtually identical to that

obtained by IRC from TS2 and which therefore has its same energy.

Furthermore, reoptimisation of the reactant was attempted by IRC from

TS1

1

and the geometry obtained substantially decreased the calculated

electronic energy barrier, from 52 kJ/mol (this number is shown in

brackets in Table 3.9) to 22 kJ/mol (see Figures 3.15 and 3.16). In this case,

the optimisations based on modified geometries allowed for a whole

reaction path to be constructed. This highlights the need for special care in

quantum cluster calculations, in which there exists a complex PES with

multiple possible pathways.

For the dimethyl substrate, the first set of barriers obtained were ΔE1 = 26

kJ/mol and ΔE2 = 14 kJ/mol. The intermediates obtained by IRC from TS1

and TS2 were different, and calculating

ΔE2 using the intermediate

obtained from TS1 yielded and erroneous value of -41 kJ/mol. As

discussed for the diethyl adduct before, this error occurs because the two

halves of the reaction path do not match. Therefore, an optimisation of TS1

was carried out with a modified geometry in which His471 was moved to

its position in TS2. The subsequent IRC calculation and optimisation of the

intermediate, however, led to a higher energy barrier for the second step

(33 kJ/mol, shown in brackets in table 3.9) than that obtained when the

intermediate is optimised from TS2 (14 kJ/mol). The barrier for the first

step of the reaction also increased, from 26 kJ/mol to 44 kJ/mol when this

new TS1 geometry was used to obtain a new reactant geometry. Therefore,

in the case of the dimethyl adduct, the geometries used to try to improve

the path led to worse results instead. The time available for this project did

not make it possible to attempt more path searches for this system, and a

continuous path is yet to be found. Yet another problem faced with the use

of restraints was that in one case, namely the dephosphorylation of the E3-

VX(R) adduct, they led to artificial ‘negative’ barriers, which means that

the geometry of the enzyme needs to relax in order to produce realistic

energies for TS and minima. For a comparison of the geometries produced

by different substrates, see Figures 3.17 – 3.20.

Figure 3.15.Effect of the position of His471 on the geometry of TS1 and reactant

on the dephosphorylation geometries of dimethyl adducts.

A. Reactant obtained

via IRC from the TS1 geometry initially optimised (magenta) and the

reactant obtained from the modified TS1 geometry (green). B. Geometry of

TS1 optimised initially (magenta) and TS1 reoptimised with a modified

geometry (see text for details) (green).

Figure 3.16.

Effect of the position of His471 on the geometry the intermediate

geometry on the dephosphorylation geometries of dimethyl adducts.A. Geometry

of the intermediate obtained via IRC from TS2 (magenta) and geometry

obtained from TS1 initially optimised (blue).

B. Geometry of the

intermediate obtained via IRC from TS2 (magenta) and geometry obtained

from TS1 reoptimised with a modified geometry (see text for details)

(green).

R1

R2

ΔE1

ΔE2

ΔE1

ΔE2

Diethyl adduct

22[52]

60

n/a

n/a

Dimethyl adduct

26[44]

14[33]

7

57

VX(R) adduct

n/d

-27

-10

n/d

VX(S) adduct

24

n/d

n/d

24

Table 3.9.

Dephosphorylation reactions with different substrates and restraints.

Electronic energy (RI-MP2/aug-cc-pvtz) barriers for the four

dephosphorylation reactions studied. Restraints were R1 (freezing the first

carbon after the functional group of a residue) or R2 (restraining all but

the substrate to the geometry already established for the reaction that has

dECP as a substrate). R2 was not applicable (n/a) to the dECP system. For

several systems transition states were not determined (n/d) as they were

not located after several attempts. All values are in kJ/mol. Numbers in

brackets indicate a less-favourable energy barrier obtained with other

geometries (as described in the text).

Figure 3.17.

Dephosphorylation reaction, reactant geometries.

A. E3-diethyl

adduct (blue) and E3-dimethyl adduct (green). B. E3-diethyl (blue) and

E3-VX(S) adduct (purple). C. Overlap of all the systems (dimethyl, diethyl

and E3-VX(S)). The geometries used in this and all other captions, unless

otherwise specified, are those that lead to a lower energy barrier, as these

would be the preferred conformations.

Figure 3.18.

Dephosphorylation reaction, TS1 geometries.

A. Diethyl (blue)

and dimethyl (green). B. Diethyl (blue) and VX(S) (purple). C. Overlap of

Figure 3.19. Dephosphorylation reaction, intermediate geometries. A. E3-diethyl

adduct (blue) and E3-dimethyl adduct (green). B. E3-diethyl (blue) and

E3-VX(R) adduct (purple). C. Overlap of these intermediate structures.

Figure 3.20. Dephosphorylation reaction, TS2 geometries. A. E3-diethyl adduct

(blue) and E3-dimethyl adduct (green). B. E3-diethyl (blue) and E3-VX(R)

adduct (purple). C. Overlap these TS2 geometries.

In document Computational studies of the E3 carboxylesterase from Lucilia cuprina (Page 89-93)