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 E32,27,28. 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 kcat/KM (106 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. Csub-Oγ Osub-NAla219 Osub-NGly136 Osub-NGly137 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 (Csub), and those between the carbonyl oxygen of the substrate (Osub) and the backbone nitrogen of given oxyanion hole In document Territorio universitario y arquitectura verde: un análisis de las presiones y las opciones sustentable para el Campus Mederos de la UANL (página 102-130)
