Inhibition by propargylglycolate substrate

In addition to being a substrate of mandelate racemase, propargylglycolate has been determined to be an inactivator of the enzyme. The process of inactivation is consistent with an enzyme-catalyzed rearrangement of the acetylenic substrate to an allenic-enol (see figure 2.9). This affords 2-keto-3-butenoate as the ultimate electrophile that can then react with an active site nucleophile resulting in inactivation of the racemase.

Figure 2.9: Scheme of the reaction of inhibition of the mandelate racemase through a michael addition reaction to the propargylglycolate derivate 2-keto-3-butenoate

In order to check the viability of this mechanism for propargylglycolate, we have calculated a reaction path from stationary point I2 (the anionic intermediate in the S to R direction) of mec II to the allenic intermediate.

This mechanism consists of three steps. First, the protonated Lys166 residue rotates to form a hydrogen bond with the Asp195 residue. This rotation step has a potential energy barrier of 2.82 kcal/mol and the formation of the new hydrogen bond stabilizes the system by 3.24 kcal/mol. In a second step, the OH group of propargylglycolate rotates from a pro-S position (with the OH group pointing to Lys166) to a pro-R position (with the OH group pointing to His297). This rotation stabilizes the system by 5.52 kcal/mol and passes over a potential energy barrier of 1.99 kcal/mol.

Figure 2.10: Energy profile for the geometric rearrangement in the active site before the inhibition by propargyl-glycolate takes place.

At this conformation of the substrate and of Lys166 (denoted I2' in figure 2.11), this same residue (the general base catalyst when the substrate is (S)-propargylglycolate) is able to protonate (S)-propargylglycolate at the C terminal position to give the allenic-enol intermediate. This proton-transfer process implies a potential energy barrier of 15.33 kcal/mol and the allenic product is stabilized by 2.77 kcal/mol with respect to I2' and 11.53 kcal/mol with respect to I2 (see figure 2.11).

The highest energy point of this QM/MM potential energy profile for the inactivation mechanism lies 17.24 kcal/mol above (S)-propargylglycolate. This energy barrier is lower than the highest energy point encountered by the system along the racemization mechanism mec II. However, it has to be remembered that we have only calculated one of the possible reaction paths from (S)-propargylglycolate to the allenic intermediate. The experimental proposal is that the formation of the allene must be faster than the rate of inactivation of the enzyme and this final covalent modification of racemase implies several more steps.

First, there is a facile ketonization of the allenic-enol intermediate to form 2-keto-3-buteonate. On our QM/MM potential energy surface, the different isomers of this buteonate molecule lie around 30 kcal/mol below the allenic intermediate. Secondly, what is properly the inactivation process consists in the attack of the electrophile molecule, via a Michaelis-type conjugate addition, to an active site nucleophile and this is the slowest part of the whole inactivation mechanism, which explains the partition ration for racemization/ inactivation of $\sim$17 000 mentioned in the the first section of this chapter.

Figure 2.11: Formation of the allenic-enol intermediate. Energies (in kcal/mol) are given with respect to stationary point I2 for mec II of propargylglycolate.