Transition-state structural refinement with GRACE and CHARMM: realistic modelling of lactate dehydrogenase using a combined quantum/classical method

Moliner, Vicent; Turner, Alexander J.; Williams, Ian H.

doi:10.1039/a702600e

Cited by 66 publications

(71 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…[48][49][50] In equation 1, HL stands for the high level chosen to obtain the corrected estimation of the activation free energy. In our case M06-2X/6-31+G(d,p) 34,35 has been used.…”

Section: Free Energy Calculationsmentioning

confidence: 99%

See 1 more Smart Citation

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

View full text Add to dashboard Cite

The promiscuous activity of the enzyme o-Succinylbenzoate Synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both Density Functional Theory and Semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylaminoacids, being N-succinyl-Rphenylglycine the best substrate. We investigated the molecular mechanisms for both reactions exploring the Potential Energy Surface. Then, Molecular Dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally-derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.

show abstract

“…[48][49][50] In equation 1, HL stands for the high level chosen to obtain the corrected estimation of the activation free energy. In our case M06-2X/6-31+G(d,p) 34,35 has been used.…”

Section: Free Energy Calculationsmentioning

confidence: 99%

“…HL calculations are carried out under the effect of the field created by the MM environment, where the electrostatic coupling between the QM and MM subsystems is calculated using point charges on the QM atoms. [48][49][50] …”

Section: Free Energy Calculationsmentioning

confidence: 99%

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

View full text Add to dashboard Cite

show abstract

“…[4][5][6][7][8] Although the relative timing of the HT and PT components is a matter of interest in mechanistic enzymology, it is not yet possible to state definitively which mechanism is preferred. 8 While a stepwise mechanism is more likely than a concerted one, results from various computational groups differ in the order in which the HT and PT steps occur.…”

mentioning

confidence: 99%

Dependence of enzyme reaction mechanism on protonation state of titratable residues and QM level description: lactate dehydrogenase

et al. 2005

Self Cite

View full text Add to dashboard Cite

We have studied the dependence of the chemical reaction mechanism of L-lactate dehydrogenase (LDH) on the protonation state of titratable residues and on the level of the quantum mechanical (QM) description by means of hybrid quantum-mechanical/molecular-mechanical (QM/MM) methods; this methodology has allowed clarification of the timing of the hydride transfer and proton transfer components that hitherto had not been possible to state definitively.It is observed macroscopically that proteins become unstable at extreme values of pH, but properties of enzymes are also pH dependent because their efficacy depends on the protonation state of their ionisable residues. The most expensive computational simulation may give meaningless results if an erroneous charge is selected for a single amino acid of the protein. Thus any theoretical analysis of protein structure or reactivity requires an adequate titration of all ionisable residues. Assignment of the protonation states of such residues is often based on the pK a of the corresponding amino acids in aqueous solution. However, this practice may introduce significant artefacts into simulations because standard pK a values of ionisable groups are shifted by local protein environments. 1 LDH is a highly stereospecific metabolic enzyme that catalyses the interconversion of pyruvate and lactate using the NADH/ NAD + pair as redox cofactor. This chemical step involves a hydride transfer (HT) from the dihydronicotinamide ring of NADH to the carbonyl C atom of pyruvate and a proton transfer (PT) to the carbonyl O atom from a N atom of the protonated His195 residue (Scheme 1).Although the rate-limiting step of the full catalytic process in the wild-type enzyme is a conformational change involving closure of a surface loop (99-110) down over the active site, 2 nonetheless the chemical step catalyzed by LDH has been extensively studied both experimentally 3 and theoretically. [4][5][6][7][8] Although the relative timing of the HT and PT components is a matter of interest in mechanistic enzymology, it is not yet possible to state definitively which mechanism is preferred. 8 While a stepwise mechanism is more likely than a concerted one, results from various computational groups differ in the order in which the HT and PT steps occur. [4][5][6][7][8] Initially the oxamate inhibitor in the ternary complex X-ray crystal structure 9 of LDH tetramer from B. stearothermophilus with NADH cofactor was replaced by pyruvate. To assign protonation states for acidic and basic residues, two alternative strategies were considered. In the first, all ionisable groups were set at a state complementary to pH 7 using the pK a values of the amino acids in aqueous solution, with the exception of His195, which was modelled in its doubly protonated form.The second protocol involved recalculation of the pK a values using the ''cluster method'', 1 as implemented by Field and coworkers, 10 according to which each titratable residue in the protein is perturbed by the electrostatic effect of the protein env...

show abstract

“…Different computational groups' proposals differ in the order in which the proton and hydride ions are transferred. Some found a mechanism in which the hydride transfer preceded proton transfer in a stepwise manner (Yadav et al 1991;Ranganathan & Gready 1997), while others predicted a concerted but asynchronous reaction in which the proton transfer was largely completed in the transition state (TS) of the reaction (Wilkie & Williams 1992;Moliner et al 1997;Turner et al 1999;Ferrer et al 2005a). A very flexible treatment of the environment (Moliner & Williams 2000) generated a potential energy surface (PES) showing the coexistence of both mechanistic pathways.…”

Section: Introductionmentioning

confidence: 99%

Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase

Ferrer

Tuñón

Moliner

et al. 2008

J. R. Soc. Interface.

View full text Add to dashboard Cite

Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the pK a value of Glu199 that becomes unprotonated at neutral pH in the mutant enzyme. This change compensates the loss of the negative charge of Asp168, rendering a still active enzyme. Thus, our methodology gives a calculated barrier height for the Asp168Ala mutant 3 kcal mol K1 higher than that for wild-type LDH, which is in very good agreement with the experiment. The computed potential energy surfaces reveal the reaction pathways and transition structures for the wild-type and mutant enzymes. Hydride transfer is less advanced and the proton transfer is more advanced in the Asp168Ala mutant than in the wild type. This approach provides a very powerful tool for the analysis of the roles of key active-site residues.

show abstract

Transition-state structural refinement with GRACE and CHARMM: realistic modelling of lactate dehydrogenase using a combined quantum/classical method

Cited by 66 publications

References 15 publications

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

Dependence of enzyme reaction mechanism on protonation state of titratable residues and QM level description: lactate dehydrogenase

Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase

Contact Info

Product

Resources

About