Reactivity for the mutant N197A

Several mutagenesis experiments on Mandelate Racemase reactivity have been reported (see page ). Some mutations on the active site show a decrease on the enzymatic reactivity rather easy to explain on the basis of the reaction mechanism that we already know (e.g. K166R, H297N). On the other hand, other mutation experiments such as the substitution of Asparagine 197 to an alanine, N197A, have provided a slight change in the racemization rate whose experimental explanation [223] is not clear from the mechanistic point of view. In figure 4.11 we show the coordination of Asn197 in the active site taken from a MD snapshot of MR wild type with mandelate substrate.

Figure 4.11: Representation of the wild type active site in a snapshot of the S reactant. Asn197 is frequently interacting with Lys166 and the substrate

The experimental work on the N197A mutation [223] points to the important role of Asn197 for the binding of intermediates analogues ($\alpha$-hydroxylbenzylphosphonate and benzohydroxamate derivatives) through an interaction with the $\alpha$-OH group of the ligand. The enzyme binds the TS analogue with affinities 100-fold greater than that observed for the substrate, while the mutant N197A binds the analogues with less affinity. In addition to the binding energy there is also a reduction of the $k_{cat}$ of 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR.

Then it is concluded that Asn197 must stabilize the TS when the racemization with the natural substrate takes place. From the chemical point of view it is not clear the stabilization interaction between the substrate and Asn197. The amide group has low capacity to withdraw electron density to stabilize the transition state, and a possible hydrogen bond between the amide group and the hydroxyl group in the substrate should not involve any important energetic change.

On the basis of the above discussion we have performed a PMF calculation on the N197A mutant for the same mechanism III step studied in the previous sections. Although the interaction substrate(QM) and Asn197(MM) should be adequately represented we assume that the non-bonded interaction already gives the tendency that should explain the mutagenesis experiment.

By circular dichroism spectroscopy it has been seen that the mutation of Asn197 to alanine does not cause any gross structural perturbation to the secondary structure[223]. Consequently, we used the same PDB coordinates that we used for the simulation in wild type enzyme. The mutation has been done manually and the setup followed the same procedure specified above for the wild type enzyme.

We have run a PMF calculation using the same reaction coordinate R$_4$ combination of the four relevant bond distances. In figure 4.12 the histogram of P(Rc) and the free energy profile are shown.

Figure 4.12: Left: the PMF profile using R$_4$ reaction coordinate. The two profiles correspond to the PMF built by WHAM or by a direct match of the overlapping windows.Right: Histogram diagram displaying the probability distribution.

Some differences appear in the PMF for the mutant N197A racemization reaction with respect to the wild type MR. As we can see in the free energy profile in figure 4.12 the S and R minimum zones have not a flat profile and the system evolves towards a more stable zone. This metastability of S and R structures is due to a new configuration of Lys166 that in wild type enzyme was not found. The flat zone representing the new free energy minimum cannot be reached by the simulation because in the new configuration Lys166 is farther from the substrate and this movement cannot be scanned by the reaction coordinate R$_4$.

There could be the possibility that the observed change is provoked by an inadequate setup instead of the mutation N197A. In order to discard this possibility a longer unconstrained molecular dynamics should be propagated. However the new configuration of Lys166 was stable during 100 ps of unconstrained molecular dynamics.

This fact would explain the lowering of enzyme activity for the mutant N197A. The change of Asn197 to Ala197 removes the possibility of the hydrogen bond between Asn197 and Lys166 as displayed in figure 4.11. This interaction was stable enough to adequately orient Lys166 in order to abstract the hydrogen in (S) configuration of the substrate. When Asn197 is removed this interaction is not present and this fact has as a consequence that Lys166 is not pointing to the substrate anymore.

We cannot energetically quantify this change because, as we said, reaction coordinate R$_4$ does not take into account the displacement of Lys166 to the new configuration. The only information that we can extract from the free energy profile in figure 4.12 is that the system just before and after the racemization reaction is found in a configuration where Lys166 is not able to abstract the proton from the substrate. A geometrical change must occur to proceed with the reaction, and the additional energy required to promote a configurational change may explain the lower rate of racemization reaction observed in the N197A mutagenesis experiment.