Xavier Prat-Resina

Microscopic pKa Analysis of Glu286 in Cytochrome c Oxidase (Rhodobacter sphaeroides): Toward a Calibrated Molecular Model†

As stringent tests for the molecular model and computational protocol, microscopic pK(a) calculations are performed for the key residue, Glu286, in cytochrome c oxidase (CcO) using a combined quantum mechanical/molecular mechanical (QM/MM) potential and a thermodynamic integration protocol. The impact of the number of water molecules in the hydrophobic cavity and protonation state of several key residues (e.g., His334, Cu(B)-bound water, and PRD(a3)) on the computed microscopic pK(a) values of Glu286 has been systematically examined. To help evaluate the systematic errors in the QM/MM-based protocol, microscopic pK(a) calculations have also been carried out for sites in a soluble protein (Asp70 in T4 lysozyme) and a better-characterized membrane protein (Asp85 in bacteriorhodopsin). Overall, the results show a significant degree of internal consistency and reproducibility that support the effectiveness of the computational framework. Although the number of water molecules in the hydrophobic cavity does not greatly influence the computed pK(a) of Glu286, the protonation states of several residues, some of which are rather far away, have more significant impacts. Adopting the standard protonation state for all titratable residues leaves a large net charge on the system and a significantly elevated pK(a) for Glu286, highlighting that any attempt to address the energetics of proton transfers in CcO at a microscopic level should carefully select the protonation state of residues, even those not in the immediate neighborhood of the active site. The calculations indirectly argue against the deprotonation of His334 for the proton pumping process, although further studies that explicitly compute its pK(a) are required for a more conclusive statement. Finally, the deprotonated Glu286 is found to be in a stable water-mediated connection with PRD(a3) for at least several nanoseconds when this presumed pumping site is protonated. This does not support the proposed role of Glu286 as a robust gating valve that prevents proton leakage, although a conclusive statement awaits a more elaborate characterization of the Glu286-PRD(a3) connectivity with free energy simulations and a protonated PRD(a3). The large sets of microscopic simulations performed here have provided useful guidance to the establishment of a meaningful molecular model and effective computational protocol for explicitly analyzing the proton transfer kinetics in CcO, which is required for answering key questions regarding the pumping function of this fascinating and complex system.

Computational study on the carboligation reaction of acetohidroxyacid synthase: New approach on the role of the HEThDP - intermediate

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl‐thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP–). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP– from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP– has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP–) and a second pyruvate molecule. Our results suggest that additional acid–base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid–base properties of the HEThDP– intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP– intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. Proteins 2010.

How important is the refinement of transition state structures in enzymatic reactions

In this paper the need to use a second derivatives direct algorithm to refine the location of transition state structures obtained in enzymatic systems has been analyzed. The 25 approximate QM/MM transition state structures previously found by means of a reaction coordinate approach for the three mechanisms of racemization of mandelate and propargylglycolate by mandelate racemase enzyme have been refined using a modified micro-iterative optimization method developed in this work. The refinement of transition state structures is especially useful to assure that a structure, found as the highest potential energy point on a profile depicted by a particular reaction coordinate, lies in the correct quadratic region. This is more important in those steps of the enzymatic process where the selected reaction coordinate may not reflect quite accurately the geometrical changes taking place in the active site.

On the modulation of the substrate activity for the racemization catalyzed by mandelate racemase enzyme. A QM/MM study

A comparative QM/MM study of the racemization reaction of two different substrates of mandelate racemase, propargylglycolate and mandelate, has been carried out. The results have been compared with those previously obtained for vinylglycolate using the same methodology. The crucial point in understanding the catalytic activity seems to be the stabilization of the anionic intermediates in the active site. Our results indicate that there are at least three different mechanisms for the racemization. It is in the third mechanism, the most favorable one, where the modulation of the rate of racemization of β,γ-unsaturated α-hydroxycarboxylates by mandelate racemase is in accord with the experimental observations. Thus mandelate is the substrate that undergoes racemization by the enzyme at the highest rate. This is due to the highest efficiency of mandelate to delocalize the extra negative charge of the transition state and the intermediate that are found along the reaction path of the α-proton abstraction. The role of propargylglycolate as an inactivator of mandelate racemase has also been studied in comparison to its activity as a substrate of the enzyme.