Phosphorylation system

Table 3.8 shows the electronic energy barriers (RI-MP2/aug-cc-pvtz) for

the phosphorylation reaction with the different substrates that were

tested. From that table, it is clear that reactions with different substrates

show very different

ΔE1 energy barriers, and that a change in the

restraints used can alter the results (or the ability to obtain them)

dramatically. The reason for the first observation is that side chain groups

can adopt slightly different positions in the presence of different

substrates, which causes large differences in the calculated energies. This

is not necessarily a problem, as such effects will also be present in the

enzyme (and is likely the reason, or part of it, why different substrates are

metabolised at different rates), but whether a cluster of this size

reproduces these effects correctly needs to be assessed (this is described in

Section 3.7). The second observation occurs because, while lack of

restraints may lead to artificial movements, an excess of restraints

prevents the system from reaching a real minimum or a first-order saddle

point. Indeed, several of these calculations did not converge after several

attempts and hundreds of hours of computer wall time used.

R1

R2

ΔE1

ΔE2

ΔE1

ΔE2

dECP

29

22

n/a

n/a

Diazinon

17

n/d

n/d

n/d

Dichlorvos

44

63[41]

31

n/d

Parathion

24

15

n/d

n/d

Table 3.8. Phosphorylation reactions with different substrates and restraints.

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

tested. 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). The

number in brackets was obtained by IRC from a modified geometry of TS1

instead of from TS2 (detailed below). R2 was not applicable (n/a) to the

dECP system. For several systems transition states were not determined

(n/d) as transition states were not located after several attempts.

It was observed that the use of restraints, especially freezing all atoms

except for the substrate, causes difficulty in locating transition states. For

this reason Table 3.8 (and also Table 3.9 in the dephosphorylation section)

contains several blank spaces that correspond to reactions for which it was

not possible to locate the transition state after many attempts. As detailed

below, at least part of the catalytic triad (the atoms that take part of the

reaction) needs to be allowed to move during optimisation, which brings

us back to the issues faced with R1.

An unexpected problem faced with R1 restraints (see Figures 3.10–3.14)

was that IRC calculations from TS1 and TS2 led in some cases to different

intermediate geometries and therefore different energies, i.e. multiple

reaction coordinates with slightly different energy barriers were possible.

This is especially true in the dephosphorylation reaction (described in the

next section). The complexity and relative flatness of the PES of a system

held together by multiple hydrogen bonds is the reason why small

changes – such as different substrates – cause the system to fall into

different energy wells in these optimisations. Examination of the

converged structures of the phosphorylation system that had dichlorvos

as a substrate (Figure 3.10), showed that in TS2 the residues His471 and

Glu351 move closer to each other in order to facilitate the transfer of a

proton between the two. This contact persists as a hydrogen bond in the

intermediate. However, this contact is non-existent in the X-ray structure

of the free enzyme and is not necessary in TS1, so it does not form during

the TS1 search and is therefore also absent in the intermediate found

following the IRC from this TS. Hence, two different intermediate

geometries with different energies are obtained from IRC of TS1 and TS2.

Changing the position of these residues in the initial structure used for

TS1 search to match the position in TS2 results in a geometry of the

intermediate (obtained by IRC) that resembles that of the intermediate

obtained by IRC from TS2 more closely than that obtained when the

residues in TS1 were not moved from the X-ray position (see Figure 3.10).

This intermediate, however, is still more than 20 kJ/mol lower in energy

than the intermediate obtained by IRC from TS2, as is evidenced by the

electronic energy barriers: 63 kJ/mol from TS2 and 41 kJ/mol from TS1 (the

number in brackets in Table 3.8). Reoptimisation of the reactant from this

modified TS1 did not cause significant changes to the energy barrier (the

difference was 2 kJ/mol).

Overlaying the different phosphorylation TS1 structures (Figure 3.12)

shows that the geometries of all systems studied present differences with

respect to dECP, although these are small in some cases. Only the

geometry of parathion differs significantly from dECP, while diazinon and

dichlorvos are a very close match. Examination of the reactant structures

shows that the reactants differ more from each other than the transition

states. Dichlorvos matches the reactant geometry of dECP better than

parathion and diazinon. These differences in geometry result in large

differences in the energy barriers seen in Table 3.8.

Figure 3.10. Various geometries of intermediate obtained in the phosphorylation

reaction of dichlorvos.

A. Intermediate geometry optimised after IRC from

TS2 (magenta) and intermediate geometry optimised after IRC from the

TS1 geometry initially found (blue). B. Intermediate geometry optimised

after IRC from TS2 (pink) and intermediate optimised from the TS2

obtained with the modified geometry in which Glu351 and His471 are at a

closer distance (green).

Figure 3.11. Phosphorylation reaction (R1 systems), reactant geometries. A. dECP (blue) and diazinon (magenta). B. dECP (blue) and

dichlorvos (green) C. dECP (blue) and parathion (purple). D. All the previous systems, overlapped. Where multiple geometries

were obtained (as described above), the geometries presented here and in all other figures are, unless otherwise specified, those

that correspond to the lowest energy barriers.

Figure 3.12. Phosphorylation reaction (R1 systems), TS1 geometries. A. dECP (blue) and diazinon (magenta). B. dECP (blue) and

dichlorvos (green) C. dECP (blue) and parathion (purple). D. Overlap of all systems.

Figure 3.13.

Phosphorylation reaction, intermediate geometries.

A. dECP (blue) and dichlorvos (green). B. dECP (blue) and parathion

(purple). C. Overlap of all systems. TS2 and intermediate were not located for diazinon.

Figure 3.14.

Phosphorylation reaction (R1 systems), TS2 geometries.

A. dECP (blue) and dichlorvos (green). B. dECP (blue) and

In document Computational studies of the E3 carboxylesterase from Lucilia cuprina (Page 83-89)