Docking organophosphates to E3 (wild type)

The binding of organophosphate pesticides to E3 is of interest as a first

step in analysing whether the blowfly can destroy other, hitherto

unstudied, compounds – in particular, warfare agents. Diazinon, dECP,

VX, and both isomers of sarin were docked into the active site cavity of

wild type E3. Furthermore, chemical warfare agents may only be tested by

a limited number of military research institutes, meaning that establishing

the potential for chemical warfare organophosphates is essential before

proceeding with experimental studies. The docking results indicate that

dECP, diazinon, sarin (both isomers) and VX(S) fit in the active and are

oriented correctly for catalysis to occur. The isomer VX(R) could not be

placed in the active site cavity in the correct orientation. The distance Oγ -

P

organophosphate

for these compounds is in general longer than those observed

for FAMEs Oγ - P

FAME

(between 3.3 and 4.2 Å). This is probably because the

leaving group of organophosphates is more bulky than those of shorter

chain FAMEs, which suggests the active site cavity may be optimised to

accommodate the latter.

The docked structure of dECP in the active site of E3 (Figure 4.6) had its

phosphorus atom 3.5 Å away from Oγ(Ser218). The phosphoryl oxygen of

dECP was placed 5.1 Å away from the backbone nitrogen of Ala219, 5.2 Å

from N(Gly136) and 3.6 Å from N(Gly137). The phosphorus atom was at a

distance of 3.5 Å from Oγ(Ser218). The position for attack by the

nucleophilic serine was good, but the phosphoryl oxygen is too far from

the oxyanion hole for hydrogen bonds to form with two of the three

residues in the hole. This is unlike the situation observed when docking

FAMEs, for which these distances were significantly shorter.

Figure 4.6. dECP docked in the active site of E3(wt). A. E3 active site cleft

and substrate. B. Close-up of dECP as docked in the active site gorge. C.

Geometry parameters of the active site. In the background Ser218 is

shown in cyan. Distances in Å.

The other pesticide tested, diazinon, was placed in the active site cavity in

a position similar to that of dECP with respect to Ser218, at a distance of

3.6 Å from Oγ. Distances from the phosphoryl sulphur to the oxyanion

holes are as follows: 5.4 Å to N(Ala219), 6.2 Å to N(Gly136), and 4.4 Å to

N(Gly137) (see Figure 4.7). These distances are longer than those observed

for FAMEs and also longer than those of dECP. This is possibly due to the

lower electronegativity of sulphur compared to oxygen.

Figure 4.7. Diazinon docked in the active site of E3(wt). A. Image of the

enzyme and active site gorge with diazinon docked.

B. Close-up of

diazinon in the gorge that leads to the active site. C. Image detailing the

geometry of the docked complex. In the background Ser218 is shown in

cyan. Distances in Å.

After the pesticides, this work focused on organophosphates poisonous to

humans such as sarin and VX. Each has two isomers, all of which were

tested. Sarin(S) (Figure 4.8) bound in a good position for attack by Ser218,

although its phosphorus is slightly distant (4.2 Å) from Oγ. This may

reflect a sub-optimal binding, yet it may be easily solved by the enzyme’s

motions accommodating the substrate in the cavity. The phosphoryl

oxygen of the substrate is 6.2 Å away from N(Ala219), 4.8 Å from

N(Gly136) and 3.1 Å from N(Gly137). Sarin(R) (Figure 4.9) was docked

with its phosphorus atom closer to Oγ (3.7 Å, compared to 4.2 Å for

sarin(S)), but it was similarly far from the oxyanion hole. The phosphoryl

oxygen was 5.8 Å away from N(Ala219), 4.8 Å from N(Gly136) and 4.2 Å

from N(Gly137).

Figure 4.8.

Sarin(S) docked in the active site of E3(wt). A. E3 active site gorge

with sarin(S) docked. B. Close-up of sarin(S) placed in the gorge (Ser218 is

shown in the background).

C. Geometry of the active site. In the

background Ser218 is shown in cyan. Distances in Å.

Figure 4.9.Sarin(R) docked in the active site of E3(wt). A. E3 active site gorge

with Sarin(R) docked. B. Close-up image of Sarin(R) placed in the cleft

that leads to the active site. C. Geometry details of the active site. In the

background Ser218 is shown in cyan. Distances in Å.

The docked structure of VX(S) had the phosphorus atom placed 3.3 Å

away from Oγ, while the phosphoryl oxygen was 4.2 Å away from

However, VX(R) (the isomer less toxic to acetylcholinesterase) could not

be placed in the active site cavity in a position and orientation suitable for

attack by Ser218. This raises the possibility that E3 may have a similar

stereoselectivity to human AChE.

Figure 4.10. VX(S) docked in the active site of E3(wt).

A. Image of the enzyme

and active site gorge with VX(S) docked. B. Close-up of VX(S) placed in

the gorge. C. Geometry detail of the active site and docked substrate. In

the background Ser218 is shown in cyan. Distances in Å.

The organophosphate compounds here analysed bind wild-type E3 in the

a productive orientation for reaction, but they do not bind the oxyanion

hole well and the phosphorus is at a longer distance from Oγ than any of

FAMEs studied. This is not surprising, as organophosphates are not the

natural substrate of E3. It was surprising, however, to find that VX(S)

docked into a position with reference parameters (distances) very similar

to those observed for FAMEs. This is the most toxic isomer of VX for

humans and animals, as it is a potent inhibitor of acetylcholinesterase

30

.

This raises the question of whether wild-type E3 can metabolise this

compound. Table 4.4 presents a summary of the distances observed for

each compound.

P

sub

-Oγ

O/S

sub

-N

Ala219

O/S

sub

-N

Gly136

O/S

sub

-N

Gly137

dECP

3.5

5.1

5.2

3.6

Diazinon

3.6

5.4

6.2

4.4

Sarin(S)

4.2

6.2

4.8

3.1

Sarin(R)

3.7

5.8

4.8

4.2

VX(S)

3.3

4.2

3.6

2.8

Table 4.4.

Key parameters of E3-organophosphate complexes.

Distances

presented are those between the reacting oxygen (Oγ) of the serine and the

reacting phosphorus atom of the substrate (P

sub

), and those between the

phosphoryl oxygen (or sulphur, in the case of diazinon) of the substrate

(O

sub

or S

sub

) and the backbone nitrogen of given oxyanion hole residues.

Distances in Å.

In document Computational studies of the E3 carboxylesterase from Lucilia cuprina (Page 120-124)