Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase

Roca, Maite; Ruiz‐Pernía, J. Javier; Castillo, Raquel; Oliva, Mónica; Moliner, Vicent

doi:10.1039/c8cp04244f

Cited by 3 publications

(6 citation statements)

References 108 publications

(187 reference statements)

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“…The resulting KIE was found to be 3.89 AE 0.15 when the ground state of BNAH in aqueous solution was used, and this parameter further increased to 4.28 AE 0.18 when the equilibrium between the Michaelis complex and the hydride transfer transition state were used for calculation (see ESI, † for details). These values are similar to those previously obtained for hydride transfer processes catalyzed by NADH-dependent enzymes, 35,36 including L-lactate dehydrogenase (in the range between 3.36 and 2.80) 37 and morphinone reductase (8.4 AE 1.6), 38 but significantly higher than that of the experimental counterpart measured in the present study. Interestingly, regarding the hydride donor-acceptor distance and the relative orientation of the different involved moieties, the nature of the optimized transition state can be considered as equivalent to those located in previous studies (Fig.…”

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confidence: 92%

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Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts

et al. 2021

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confidence: 92%

“…3). [35][36][37][38] It therefore suggests that the hydride transfer step in the T-Sav artificial enzyme is at least partially rate-limiting.…”

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confidence: 99%

Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts

et al. 2021

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“…Computational studies of FDH have identified important interactions in the active site, first, in classical simulations by Bruice 36 and, later, in QM/MM studies by Popov. 35 Freeenergy calculations by Moliner, 37 with the use of the AM1 semiempirical Hamiltonian, found a free-energy barrier equal to 12.1 kcal/mol.…”

Section: ■ Introductionmentioning

confidence: 99%

Role of Protein Motions in Catalysis by Formate Dehydrogenase

Antoniou

Schwartz

2020

J. Phys. Chem. B

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We have analyzed the reaction catalyzed by formate dehydrogenase using transition path sampling. This system has recently received experimental attention using infrared spectroscopy and heavy-enzyme studies. Some of the experimental results point to the possible importance of protein motions that are coupled to the chemical step. We found that the residue Val123 that lies behind the nicotinamide ring occasionally comes into van der Waals contact with the acceptor and that in all reactive trajectories, the barrier-crossing event is preceded by this contact, meaning that the motion of Val123 is part of the reaction coordinate. Experimental results have been interpreted with a two-dimensional formula for the chemical rate, which cannot capture effects such as the one we describe.

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“…In Tyr196AzAla a VET pathway along NAD + is highly probable, which requires efficient VET between three molecules, from FDH to NAD + and from NAD + to N − 3 . In the previous study on Pseudomonas FDH, motions of various active-site residues were identified as integral part of the reaction coordinate [263]. Because of the high sequential and structural similarity to Cb FDH, these residues can clearly be identified as Val93, 1 Because of the lack of mutant structures, distances are measured from the C β of the substituted amino acid.…”

Section: Resultsmentioning

confidence: 99%

“…One study focused on the KIE of FDH WT (based on the structure of Pseudomonas sp. FDH) and on the interpretation of the KIE in the context of enzymatic reactions [263]. The other study was building on the effects observed from Val123 mutation [225].…”

Section: Low-frequency Oscillations In Formate Dehydrogenasementioning

confidence: 99%

Protein vibrational energy transfer: new sensors and application in an enzyme active site

Löffler

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Experiments on Vibrational Energy Transfer (VET) in proteins contribute to our understanding of fundamental biological processes such as allostery, dissipation of excess energy, and possibly enzymatic catalysis. While these processes have been studied for a long time, many questions remain unanswered. The aim of this work was to expand the application of existing spectroscopic techniques to investigate VET, seeking tailored solutions for the diversity of proteins and amino acid environments. Additionally, new target proteins were to be established to broaden the spectrum of VET experiments towards the role of VET and low-frequency protein modes (LFMs). To test their suitability as VET sensors, the non-canonical amino acids (ncAAs) Azidoalanine (N3Ala), azido-L-Homoalanine (Aha), p-azido-Phenylalanine (N3Phe), p-cyano-Phenylalanine (CNPhe), and 4-cyano-Tryptophan (CNTrp) were coupled to the VET donor β-(1-azulenyl)-L-Alanine (AzAla) in dipeptides. Their spectral properties were compared using FTIR and VET spectra in H2O, dimethyl sulfoxide, and tetrahydrofuran. The solvent strongly influences the measured VET signals, which can be explained by the direct interaction of the solvent with the dipeptides. Additionally, the peak time within the subgroups of azide and nitrile sensors increased with the size of the side chain, indicating the dependence between peak time and the distance between VET donor and sensor. When incorporated into a protein, solvent interactions are less dominant. Therefore, Aha, N3Phe, and CNPhe were additionally incorporated at two different positions in the PDZ protein domain and investigated. Due to Fermi resonances, signals from azide sensors are challenging to predict, unlike those of the nitrile sensors. Overall, the experiments showed that nitrile groups can serve well as VET sensors, as their lower extinction coefficient is compensated for by a narrower bandwidth. This expands the number of potential target proteins, and sensor incorporation can be less disruptive at various protein locations. Since the VET donor AzAla can inject the energy of a photon into a protein as vibrational energy at a specific location, it can also be used for the targeted excitation of LFMs. If these modes are involved in an enzymatic reaction, a direct influence on activity is expected. This hypothesis has long existed but has not been definitively verified. Some studies have found evidence for the involvement of LFMs in formate dehydrogenase (FDH) catalysis. Therefore, FDH was chosen for the investigation of LFMs in enzymes. This specific system additionally allows the use of a natural VET sensor: it forms a stable complex with NAD+ and N3-, an excellent IR marker. Thus, it provided the opportunity to test low-molecular-weight non-covalent ligands as VET sensors. After ensuring sufficient AzAla supply through the internal establishment of an enzymatic synthesis, AzAla could be incorporated at various positions in FDH. Despite spectral overlap between free and bound N3-, the latter could be identified by its narrower FWHM. For some variants, no binding could be observed. Circular dichroism spectra showed that these variants structurally deviate slightly from other variants and the wild type (WT). VET could be observed over 22 Å from two regions of the protein to the N3- bound in the active center, at protein concentrations of below 2 mM. Unbound N3- did not generate signals, allowing it to be added in excess ensuring the saturation of the protein in VET experiments. The activity of FDH WT and four AzAla mutants was investigated under substrate saturation without and with AzAla excitation. In these experiments, a slight reduction in activity under illumination was observed, even for the WT, who is not expected to interact with the excitation light. So far, a difference in sample temperature cannot be excluded as the cause for this decline. The presented experiments with FDH illustrate the potential of low-molecular-weight ligands as VET sensors, with N3- being particularly attractive due to its simple structure (preventing Fermi resonances) and its high extinction coefficient. Its use can add many metalloproteins as potential targets for VET experiments and allows investigation without a VET sensor ncAA. Additionally, initial experiments were conducted to measure light-dependent FDH activity. By specifically exciting protein LFMs, this project could contribute in the future to answering longstanding questions about the extraordinary catalytic efficiency of enzymes.

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Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase

Abstract: The protein cannot be considered as a passive spectator of the chemical system; it is part of the chemical reaction.

Cited by 3 publications

References 108 publications

Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts

Transfer hydrogenations catalyzed by streptavidin-hosted secondary amine organocatalysts

Role of Protein Motions in Catalysis by Formate Dehydrogenase

Protein vibrational energy transfer: new sensors and application in an enzyme active site

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