Docking FAMEs to E3 (wild type)

Fatty acid methyl esters (FAMEs) are some of the most plausible

substrates for E3, given their co-location in fat bodies and the fact that they

are carboxylesters, which is known to be the broad substrate class for

E3

2,27,28

_{. In this section, the results of docking different FAMES to E3 (wild}

type) to test the possibility of a productive binding to the active site are

presented. A series of potential fatty acid methyl ester substrates were

docked into the active site of E3 using Autodock Vina. Each docking run

produced several poses: only the lowest energy (i.e. most stable) poses,

which were assessed by visual inspection, are discussed here.

Examination of the enzyme:substrate complexes shows that all substrates

are coordinated in a way that positions them correctly for nucleophilic

attack by Ser218 of the catalytic triad of E3. Five FAMEs of different length

were investigated: methyl hexanoate (6C), methyl octanoate (8C), methyl

decanoate (10C), methyl laurate (12C) and methyl myristate (14C). In each

case, the carbonyl carbon atom of the FAME is between 3.4 and 3.6 Å from

the nucleophilic Oγ of Ser218, a distance that indicates the substrate is well

positioned for attack (for geometry parameters see Table 4.3). FAMEs with

a long chain are positioned with it folded rather than extended along the

active site gorge, most likely to shield it from polar solvent.

Methyl hexanoate is positioned within the active site with its short (6C)

chain along the active site cleft. Its carbonyl carbon is positioned 3.5 Å

away from Ser218-Oγ, which is an appropriate distance for approach and

attack by Ser218. It is also well positioned with respect to the oxyanion

hole, having its carbonyl oxygen pointing towards it at a distance of 4.5 Å

from the backbone nitrogen of Ala219, 3.0 Å from N(G136) and 3.2 Å from

N(G137) (Figure 4.1).

Figure 4.1. Methyl hexanoate docked in the active site of E3(wt). A. View of

the active site gorge of E3, with methyl hexanoate lying in it. B. Close-up

of the gorge, which shows the chain of methyl hexanoate extended along

it. In the background Ser218 is shown in cyan. C. Detail of the active site

with the substrate placed in it. Distances in Å. Hydrogen atoms have

been omitted for clarity.

Methyl octanoate (Figure 4.2) was docked into the active site in a position

very similar to that of methyl hexanoate. The carbonyl carbon is 3.4 Å

away from the hydroxyl oxygen of Ser218, while the carbonyl oxygen is 4.8

Å away from N(Ala219), 4.3 Å from N(Gly136) and 3.2 Å from N(Gly137).

Figure 4.2.Methyl octanoate docked in the active site of E3(wt).A. View of the

active site gorge of E3, with methyl octanoate lying in it. B. Close-up of

the gorge, which shows the chain of methyl octanoate extended along it.

In the background Ser218 is shown in cyan. C. Detail of the active site

with methyl octanoate in it. Distances in Å. Hydrogen atoms have been

omitted for clarity.

Methyl decanoate was also positioned within the active site in a good

orientation for attack, with its carbonyl carbon 3.5 Å away from Oγ(see

Figure 4.3). The distances between the carbonyl oxygen of the substrate

and the oxyanion hole are as follows: 4.5 Å from N(Ala219), 3.0 Å from

N(Gly136) and 3.2 Å from N(Gly137). Its chain lies in a folded

conformation rather than extended along the cleft.

Figure 4.3.

Methyl decanoate docked in the active site of E3(wt). A. Image of

E3 that shows the active site cleft and methyl decanoate in it. B. Close-up

of the cleft and substrate.

C. Image of the active site with methyl

decanoate docked. Distances in Å. All hydrogen atoms are omitted for

clarity. In the background Ser218 is shown in cyan.

The long chain (12C) of methyl laurate is also folded like in the case of

methyl decanoate, rather than extended along the cleft. The position of

methyl laurate in the active site is similar to that of the previous

substrates, in which the carbonyl oxygen is tilted towards the members of

the oxyanion hole. The distances are as follows: 4.5 Å to N(Ala219), 3.1 Å

to N(Gly136), and 3.2 Å to N(Gly137). The carbonyl carbon is 3.5 Å away

Figure 4.4. Methyl laurate docked in the active site of E3(wt). A. Image of the

enzyme where the gorge and substrate are visible. B. Closer image of

methyl laurate positioned in the active site and gorge (Ser218 shown in

magenta). C. Active site and substrate in detail. Distances in Å. Hydrogens

are omitted for clarity. In the background Ser218 is shown in cyan.

Methyl myristate also has a long chain (14C) that lies in a folded

conformation in the active site cleft (Figure 4.5). Its carbonyl group lies 3.7

Å away from Oγ (a bit further than the other FAMEs that were tested). The

carbonyl oxygen is 4.5 Å away from N(A219), 4.1 Å from N(Gly136) and

3.1 Å from N(Gly137). Overall, the position of methyl myristate is good for

attack but less optimal than that of the other FAMEs discussed here.

Figure 4.5. Methyl myristate docked in the active site of E3(wt).

A. E3 active

site cleft and substrate. B. Close-up of methyl myristate positioned in the

active site and gorge. C. Geometry details of the active site. Distances in

Å. All hydrogens are omitted for clarity.

All the FAMEs studied were docked in the active site of wild-type E3 in

the correct position for attack. The position of the carboxyl centre with

respect to the catalytic triad and oxyanion hole was very similar for all

substrates, only methyl myristate was placed a bit further from these

centres than the other substrates but still in a good position. It is clear that

all the FAMEs tested fit correctly in the active site cavity. Experimental

data taken by Mr Faisal Younis at the CSIRO shows that catalytic rates are

very different for different FAMEs (see Table 4.2). Although substrate

binding parameters, such as distances and angles, are not enough to

predict reaction kinetics, docking techniques have been useful in showing

that all these compounds can bind in the right conformation to undergo

attack, and to analyse how FAMEs are positioned with respect to the active

site groups. This information will be used in the next section to compare to

how non-physiological substrates bind E3. The structures obtained by

docking organophosphates to E3 were used as a starting point for MD

simulations (presented later in this chapter). Unfortunately, time

constraints did not allow for simulations of E3 with FAMEs bound to be

performed, and so the comparison of natural and unnatural substrates will

be part of another project.

Substrate

k

cat

/K

M

(10

6

M

-1

s

-1

)

Methyl hexanoate

0.28 ± 0.02

Methyl octanoate

0.83 ± 0.07

Methyl decanoate

1.38 ± 0.02

Methyl laurate

0.2 ± 0.03

Methyl myristate

0.061 ± 0.001

Diethyl 4-methylumbelliferyl phosphate

0.05 ± 0.005

Table 4.2. Kinetic parameters of substrate hydrolysis by E3. Data from Ref. 29.

C

sub

-Oγ

O

sub

-N

Ala219

O

sub

-N

Gly136

O

sub

-N

Gly137

Methyl hexanoate

3.5

4.5

3.0

3.2

Methyl octanoate

3.4

4.8

4.3

3.2

Methyl decanoate

3.5

4.5

3.0

3.2

Methyl laurate

3.5

4.5

3.1

3.2

Methyl myristate

3.7

4.5

4.1

3.1

Table 4.3. Key parameters of the E3-FAME complexes. Distances presented are

those between the reacting oxygen (Oγ) of the serine and the carbonyl

carbon of the substrate (C

sub

), and those between the carbonyl oxygen of

the substrate (O

sub

) and the backbone nitrogen of given oxyanion hole

In document Computational studies of the E3 carboxylesterase from Lucilia cuprina (Page 115-120)