Reaction mechanism of propargylglycolate substrate

As mentioned previously three different reaction mechanisms were found for vinylglycolate in the previous paper[212]. Mec I and mec II start from the same structure, which we have called structure S. However, mec III starts from a different structure denoted S2. The corresponding structures for propargylglycolate, S and S2, have also been found here (see figure 2.5) by substituting vinylglycolate by propargylglycolate in each case and optimizing the moving part of the system.

The labels used in figure 2.5 to identify some atoms will be used throughout the chapter and correspond to atoms located in eight positions that are able to be occupied or unoccupied by means of proton transfer processes. It should be taken into account that positions 1 and 7 are really the same, but when the hydrogen is attached to C1 or C7 it means that the proton comes from the pro-(S) or from the pro-(R) side, respectively.

(S) structure:
A comparison of these structures with the corresponding S and S2 geometries of vinylglycolate does not reveal any significant differences. In all the structures Lys166 residue is closer to the substrate than His297. This fact is consistent with the basic catalytic role attributed to Lys166 residue in the S-to-R direction. His297 residue is nearly 1.0 Å closer to the substrate in S2 than in S (the distances from the hydrogen atom attached to N2 to C1 are 2.95 Å and 3.85 Å, respectively). This is a very important geometric difference that will have mechanistic consequences, as will be described later.

There are two other structural differences related to the location of Lys166 and His297 residues in the active site of the minimum energy structures of S and S2. First, whereas in S2 Lys166 forms a hydrogen bond with one of the ligands of magnesium ion (Asp195), in S Lys166 and Asp195 residues are quite far away. Secondly, a hydrogen bond between His297 and Glu247 exists in S but it does not form in S2. On the other hand, the hydrogen bond between propargylglycolate and Lys164 is present in the two minimized structures S and S2. In both cases, the distance (2.70 Å in S and 2.69 Å in S2) between the heavy atoms (O4-N3) involved in this hydrogen bond, compares well with the X-ray experimental value of 2.76 Å [233].

Finally, a hydrogen bond between the substrate and Glu317 residue appears in S as well as in S2. However, the O-O distance in this hydrogen bond is slightly longer for S2 than for S. These two bond distances are in good agreement with the experimental result of 2.68 Å [233].

Figure 2.5: (a) Structure of the active site at the stationary point S for propargylglycolate. The labels used correspond to the following atoms: C1, $\alpha$-carbon of (S)-propargylglycolate enantiomer; N2, $\epsilon$-nitrogen of His297; N3, amino nitrogen of Lys164; O4, oxygen of the carboxylate group of the substrate; O5, oxygen of the carboxylic group of Glu317; O6, oxygen of the carboxylate group of the substrate; N8, amino nitrogen of Lys166. (b) Structure of the active site at the stationary point R for propargylglycolate. The labels used to identify some atoms are the same as in (a), with the exception of C7, that corresponds to the $\alpha$-carbon of (R)-propargylglycolate enantiomer. (c) Structure of the active site at the stationary point S2 for propargylglycolate. Distances are given in Å.

a$)$
b$)$
c$)$

(R) structure:
The minimization of the QM/MM potential energy starting from the minimum energy structure of (R)-vinylglycolate and Mandelate Racemase enzyme but substituting vinylglycolate by propargylglycolate, leads to the minimum energy structure partially represented in 2.5 (structure R). Contrary to structures S and S2, the deprotonated $\epsilon$-nitrogen of His297 is oriented to the $\alpha$-proton attached to the C7 carbon forming with it a hydrogen bond of 1.76 Å. This is the expected orientation of His297 if it is supposed to be the basic catalyst in the R$\to$S direction. In this R structure His297 does not interact with Glu247. In addition, we can observe how the conjugate acid of Lys166 has moved further from the $\alpha$-carbon with respect to its position in structure S, and it is interacting (as in S2) with a magnesium ligand (Asp195) by hydrogen bonding. The two other hydrogen bonds between propargylglycolate and the two residues Lys164 and Glu317 are still maintained in the active site of the (R)-enantiomer.

Mechanism I and II for propargylglycolate:
For propargylglycolate mec I and mec II start from the same structure S and consist of six steps that are qualitatively very similar to the case of vinylglycolate. Figure 2.6 and 2.7 show the energy profile of mechanism I and II, respectively. In these two mechanisms, a proton transfer (which we will denote ``catalytic'' from now on) precedes the proton transfer corresponding to the racemization process itself (which we will denote ``reactive'' from now on). The role of those catalytic steps is to diminish the negative charge on the substrate along the reaction. In mec I the catalytic proton transfer takes place from the $\epsilon$-ammonium group of Lys164 to the O4 of the substrate. In mec II it is through the hydrogen bond between Glu317 and one of the carboxylic oxygens (O6) of the substrate. The result of this step (S $\rightarrow$ I1) in both mechanisms is the neutral substrate, that is propargyl acid within our model. The catalytic proton transfer in mec I presents a higher potential energy barrier (15.9 kcal/mol) than in mec II (11.8 kcal/mol). In addition, the product of this proton transfer, intermediate I1, is around 6 kcal/mol more endoergic than S in mec I, whereas S and I1 are nearly isoergic in mec II. The catalytic proton transfers do not provoke significant changes in the bond distances involved in the reactive proton transfers of mec I and mec II.

Figure 2.6: Potential energy profile for the mechanism I of propargylglycolate substrate racemization

Figure 2.7: Potential energy profile for the mechanism II of propargylglycolate substrate racemization

Step 2 in both mechanisms corresponds to the abstraction of the $\alpha$-proton attached to C1 by Lys166, that is the first reactive proton transfer of the racemization process, and leads in each case to the anionic structures I2. Parallel to this proton transfer, residue Lys166 forms a new hydrogen bond with Asp195, one of the ligands of the magnesium ion. The other reactive bond distance N2-H remains practically unaltered from I1 to I2 whereas the reactive bond distance C7-H shows a slight decrease, indicating a small approximation of residue His297 to the substrate. In any case, at I2 His297 (the residue that is the acid catalyst in the S$\to$R direction) is still quite far from propargylglycolate. Steps 3 and 4 do not correspond to any proton-transfer process. The energy barriers of these two steps are the result of some important changes in the active site. First, residue His297 approaches the substrate and the C7-H distance clearly diminishes (from 3.70 Å at I2 to 1.71 Å at I4 in mec I and from 3.67 Å at I2 to 1.80 Å at I4 in mec II). Secondly, parallel to its approximation to propargylglycolate, residue His297 loses its hydrogen-bond interaction with Glu247 and, in consequence, the N2-H$\cdots$Glu247 bond distance increases to 2.94 Å and to 2.96 Å at I4 in mec I and mec II, respectively. Thirdly, the configuration change of C1: from a configuration closer to that of the reactant S to a configuration similar to that of R, through an sp2 hybridization. That is, the change from C1 to C7 in our code. In both mechanisms this configuration change takes place in step 4 and involves the energy maxima of the reaction paths. This change is produced when His297 is close enough to the anionic intermediate to form an ionic pair with it.

Step 5 is the second reactive step that consists of the proton transfer from N2 to C7, through structure TS5. From I4 this step presents a smaller barrier than the first reactive proton transfer and is exoergic in mec I or nearly isoergic for mec II. The final product R is obtained in both mechanisms after the catalytic proton transfer during step 6, that returns the catalytic positions to their initial state.

Mechanism III for propargylglycolate: 
  As described in the previous paper of the group [212] mec III for vinylglycolate is clearly different from mec I and mec II. This alternative mechanism has also been found for propargylglycolate in MR as a third feasible reaction path of racemization. Mec III starts from the structure denoted S2 (see figure 2.5).

In the case of vinylglycolate this mechanism, much simpler than the two previous reaction paths, was shown to take place, in only two steps (without any catalytic proton transfer) via a dianionic intermediate I^3.8. The stabilization of this intermediate is achieved in mec III by the residue His297, which from the beginning (in S2) is situated closer to the substrate than in S. The two steps of this mechanism consist in part in the two reactive proton transfers. The first between positions C1 and N8 (Lys166) through TS1 and the second between positions C7 and N2 (His297) through TS2. In a concerted manner with the $\alpha$-proton abstraction by Lys166, the configuration change of C1 and the approach of His297 take place. Therefore, the so-called intermediate between TS1 and TS2 is a product-like structure, with a C7 clearly in R configuration rather than the planar sp2 hibridisation structure.

In contrast to the results for vinylglycolate, where the two-step mechanism was found, mec III for propargylglycolate + MR is a one-step mechanism with only one clear energetic barrier of 21.9 kcal/mol at TS1. The reaction coordinate corresponding to this mec III from S2 to R was recalculated with a smaller step trying to find a minimum energy structure for the dianionic intermediate I of propargylglycolate. Even so and although the reaction energy profile shows a plateau in between 1.65 Å and 1.58 Å for the C7-H bond, no minimum was located with the L-BFGS minimizer.