Juan Bertrán

Theoretical insights in enzyme catalysis

In this tutorial review we show how the methods and techniques of computational chemistry have been applied to the understanding of the physical basis of the rate enhancement of chemical reactions by enzymes. This is to answer the question: Why is the activation free energy in enzyme catalysed reactions smaller than the activation free energy observed in solution? Two important points of view are presented: Transition State (TS) theories and Michaelis Complex (MC) theories. After reviewing some of the most popular computational methods employed, we analyse two particular enzymatic reactions: the conversion of chorismate to prephenate catalysed by Bacillus subtilis chorismate mutase, and a methyl transfer from S-adenosylmethionine to catecholate catalysed by catechol O-methyltransferase. The results and conclusions obtained by different authors on these two systems, supporting either TS stabilisation or substrate preorganization, are presented and compared. Finally we try to give a unified view, where a preorganized enzyme active site, prepared to stabilise the TS, also favours those reactive conformations geometrically closer to the TS.

Bidimensional tunneling dynamics of malonaldehyde and hydrogenoxalate anion. A comparative study

One‐dimensional and bidimensional tunneling splittings have been calculated in malonaldehyde (MA) and hydrogenoxalate anion (HX) systems. Two different monodimensional paths have been considered: the intrinsic reaction path (IRP) and the linear reaction path (LRP). A bidimensional model that includes the coupling between the proton transfer motion and the vibration of the heavy atoms is then used. We find that with the bidimensional model the splittings are 2 orders of magnitude greater than the monodimensional ones, and close to the previous experimental and theoretical values for the MA when zero point energy is introduced. At all levels of calculation we obtain that the splitting is greater in the MA than in the HX. This fact is attributed to the different size of the rings through which the proton transfer occurs.

Molecular electric properties and nuclear and vibrational relaxation. An ab initio study of HF, CH4 and C2H4

The effects of nuclear relaxation and change in vibrational molecular energy are accounted for in the theoretical determination of the molecular properties of HF, CH4, and C2H4. A method based on a finite-difference technique is employed. An analysis of the influence of these contributions versus the purely electronic contribution is made, together with an appraisal of the effect of vibrational excitation.

Protonation of glycine, serine and cysteine. Conformations, proton affinities and intrinsic basicities

Abstract Proton affinities and gas-phase basicities of glycine, serine and cysteine have been computed using the three-parameter B3LYP density functional approach. For that, the geometry and vibrational frequencies of several conformations of neutral and protonated glycine, serine and cysteine have been explored. The preferred site for protonation in all aminoacids is the amino group. The lowest conformation always shows an intramolecular hydrogen bond between NH3+ and the carbonylic oxygen. For serine and cysteine, additional hydrogen bonds may be formed, the favored interaction being that in which the oxygen or sulfur atoms of the side chain interact, as proton acceptor, with NH3+. The computed B3LYP proton affinities and gas-phase basicities are in very good agreement with the known experimental data.

Promiscuity in Alkaline Phosphatase Superfamily. Unraveling Evolution through Molecular Simulations

We here present a theoretical study of the alkaline hydrolysis of a phosphodiester (methyl p-nitrophenyl phosphate or MpNPP) in the active site of Escherichia coli alkaline phosphatase (AP), a monoesterase that also presents promiscuous activity as a diesterase. The analysis of our simulations, carried out by means of molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials, shows that the reaction takes place through a D(N)A(N) or dissociative mechanism, the same mechanism employed by AP in the hydrolysis of monoesters. The promiscuous activity observed in this superfamily can be then explained on the basis of a conserved reaction mechanism. According to our simulations the specialization in the hydrolysis of phosphomonoesters or phosphodiesters, developed in different members of the superfamily, is a consequence of the interactions established between the protein and the oxygen atoms of the phosphate group and, in particular, with the oxygen atom that bears the additional alkyl group when the substrate is a diester. A water molecule, belonging to the coordination shell of the Mg(2+) ion, and residue Lys328 seem to play decisive roles stabilizing a phosphomonoester substrate, but the latter contributes to increase the energy barrier for the hydrolysis of phosphodiesters. Then, mutations affecting the nature or positioning of Lys328 lead to an increased diesterase activity in AP. Finally, the capacity of this enzymatic family to catalyze the reaction of phosphoesters having different leaving groups, or substrate promiscuity, is explained by the ability of the enzyme to stabilize different charge distributions in the leaving group using different interactions involving either one of the zinc centers or residues placed on the outer side of the catalytic site.

Analytical energy derivatives for a realistic continuum model of solvation: Application to the analysis of solvent effects on reaction paths

Analytical expressions for the first and second derivatives of the Hartree–Fock energy have been derived in case of a solvated system simulated by a multipolar charge distribution embedded in a cavity of arbitrary shape and a solvent represented by a dielectric continuum. A computer code has been written on these bases. It allows geometry optimizations and more generally the determination of the critical points of the potential energy surface for a molecular system interacting with a solvent as easily as in the case of an isolated molecule. The use of this code is illustrated by the computation of the main features of the reaction path of a Menshutkin‐type reaction in various solvents. The results compare pretty well with those obtained by a full Monte Carlo simulation of the solvent by Gao. This agreement supports the idea that solvents, including water, can be safely modeled by a continuum. The advantage of such models rests in the fact that they allow refined computations on the solute at a minimum com...

Theoretical Study of Phosphodiester Hydrolysis in Nucleotide Pyrophosphatase/Phosphodiesterase. Environmental Effects on the Reaction Mechanism

We here present a theoretical study of the alkaline hydrolysis of methyl p-nitrophenyl phosphate (MpNPP(-)) in aqueous solution and in the active site of nucleotide pyrophosphatase/phosphodiesterase (NPP). The analysis of our simulations, carried out by means of hybrid quantum mechanics/molecular mechanics (QM/MM) methods, shows that the reaction takes place through different reaction mechanisms depending on the environment. Thus, while in aqueous solution the reaction occurs by means of an A(N)D(N) mechanism, the enzymatic process takes place through a D(N)A(N) mechanism. In the first case, we found associative transition-state (TS) structures, while in the enzyme TS structures have dissociative character. The reason for this change is rationalized in terms of the very different nature of the electrostatic interactions established in each of the environments: while the aqueous solution reduces the repulsion between the negatively charged reacting fragments, assisting their approach, the NPP active site stabilizes the charge distribution of dissociative TS structures, allowing the reaction to proceed with a significantly reduced free energy cost. Interestingly, the NPP active site is able to accommodate different substrates, and it seems that the nature of the TSs depends on their electronic characteristics. So, in the case of the MpNPP(-) substrate, the nitro group establishes hydrogen-bond interactions with water molecules and residues found in the outer part of the catalytic site, while the leaving group oxygen atom does not coordinate directly with any of the zinc atoms of the active site. If methyl phenyl phosphate is used as substrate, then the charge on the leaving group is supported to larger extent by the oxygen atom and the phenolate anion can be then coordinated to one of the two zinc atoms present in the active site.

Cu2+/+ Cation Coordination to Adenine-Thymine Base Pair. Effects on Intermolecular Proton-Transfer Processes

Intermolecular proton-transfer processes in the Watson & Crick adenine-thymine Cu+ and Cu2+ cationized base pairs have been studied using the density functional theory (DFT) methods. Cationized systems subject to study are those resulting from cation coordination to the main basic sites of the base pair, N7 and N3 of adenine and O2 of thymine. For Cu+ coordinated to N7 or N3 of adenine, only the double proton-transferred product is found to be stable, similarly to the neutral system. However, when Cu+ interacts with thymine, through the O2 carbonyl atom, the single proton transfer from thymine to adenine becomes thermodynamically spontaneous, and thus rare forms of the DNA bases may spontaneously appear. For Cu2+ cation, important effects on proton-transfer processes appear due to oxidation of the base pair, which stabilizes the different single proton-transfer products. Results for hydrated systems show that the presence of the water molecules interacting with the metal cation (and their mode of coordination) can strongly influence the ability of Cu2+ to induce oxidation on the base pair.

Theoretical modeling of the reaction mechanism of phosphate monoester hydrolysis in alkaline phosphatase.

The reaction mechanism of phosphate monoester hydrolysis in alkaline phosphatase is analyzed by means of hybrid QM/MM simulations. A recently developed semiempirical Hamiltonian, AM1/d-PhoT, which takes into account the d orbitals on the phosphorus atom, has been employed. The reaction mechanism obtained is either associative or dissociative, depending on the size of the QM subsystem. The results are rationalized on the basis of the degree of charge transfer from the reacting fragments to the two zinc ions present in the active site, which has been observed to be dependent on whether or not metal atoms and their coordination spheres are included in the QM region. The description obtained using the largest QM region agrees with the picture obtained from experimental data.

Theoretical study of reaction mechanisms for the ketonization of vinyl alcohol in gas phase and aqueous solution

Theoretical ab initio calculations are done on different mechanisms for the conversion of vinyl alcohol to acetaldehyde, both in gas phase and in solution. Several basis sets are used in order to assess the accuracy of the results in gas phase and a continuum model of the solvent is employed to mimic reactions in water solution. The results indicate a catalytic action of water in hydrated clusters in gas phase, whereas in solution, and within the error limits of our calculations, both neutral water-chain and ionic mechanisms appear to be equally probable. Finally, the action of acids or bases is tested through the analysis of the reaction of vinyl alcohol with H3O+ and HO−. The results of the calculations are shown to be in qualitative agreement with the experimental facts when 6-31++G basis set is used but not when either STO-3G or 4-31G basis sets are employed.