Direct Proton Transfer Research Articles

The mechanism of the NHC catalyzed aza-Morita–Baylis–Hillman reaction: insights into a new substrate-catalyzed bimolecular pathway

The first mechanistic study on the NHC-catalyzed aza-MBH reaction between cyclopentenone and N-mesylbenzaldimine using density functional theory reveals that a bimolecular mechanism, involving two molecules of benzaldimine in the proton transfer, is energetically more preferred over the conventional direct proton transfer.

Fundamental Reaction Pathway for Peptide Metabolism by Proteasome: Insights from First-Principles Quantum Mechanical/Molecular Mechanical Free Energy Calculations

Proteasome is the major component of the crucial non-lysosomal protein degradation pathway in the cells, but the detailed reaction pathway is unclear. In this study, first-principles quantum mechanical/molecular mechanical free energy calculations have been performed to explore, for the first time, possible reaction pathways for proteasomal proteolysis/hydrolysis of a representative peptide, succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC). The computational results reveal that the most favorable reaction pathway consists of six steps. The first is a water-assisted proton transfer within proteasome, activating Thr1-O(γ). The second is a nucleophilic attack on the carbonyl carbon of a Tyr residue of substrate by the negatively charged Thr1-O(γ), followed by the dissociation of the amine AMC (third step). The fourth step is a nucleophilic attack on the carbonyl carbon of the Tyr residue of substrate by a water molecule, accompanied by a proton transfer from the water molecule to Thr1-N(z). Then, Suc-LLVY is dissociated (fifth step), and Thr1 is regenerated via a direct proton transfer from Thr1-N(z) to Thr1-O(γ). According to the calculated energetic results, the overall reaction energy barrier of the proteasomal hydrolysis is associated with the transition state (TS3(b)) for the third step involving a water-assisted proton transfer. The determined most favorable reaction pathway and the rate-determining step have provided a reasonable interpretation of the reported experimental observations concerning the substituent and isotopic effects on the kinetics. The calculated overall free energy barrier of 18.2 kcal/mol is close to the experimentally derived activation free energy of ∼18.3-19.4 kcal/mol, suggesting that the computational results are reasonable.

Improvement of biohydrogen generation and seawater desalination in a microbial electrodialysis cell by installing the direct proton transfer pathway between the anode and cathode chambers

We are focusing on the enhancement of microbial electrodialysis cell (MEDC) performance by alleviation of pH gradient between the anode and cathode chambers by setting up a direct proton transfer pathway, which allows protons to migration unconstrainedly, with three different membranes ultrafiltration membrane (UF), anion-exchange membrane (AEM), cation-exchange membrane (CEM)) in the MEDC. Setting up a direct proton transfer pathway between the anode and cathode chamber in the MEDC abated pH gradient by up to about 54%. Also, hydrogen production and salt removal efficiency were enhanced. In a comparison of membranes for a direct proton transfer pathway, an AEM has the best performance for reduction for pH gradient because of a higher proton transfer by phosphate anions, but due to the high substrate permeability of an AEM, the hydrogen production with AEM was lower than that with UF—which highest hydrogen production was observed with UF (5.77±0.54 mL, 0.55±0.14 mL/h). In terms of salt removal efficiency, using CEM as a direct proton transfer pathway showed the highest performance (77.63%)

PH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis)

Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-d-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9±0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation.

Multiple Mechanisms in Pd(II)-Catalyzed SN2′ Reactions of Allylic Alcohols

Density functional calculations and experiments were used to examine mechanisms of Pd(II) catalyzed intramolecular cyclization and dehydration in acyclic and bicyclic monoallylic diols, a formal S(N)2' reaction. In contrast to the previously proposed syn-oxypalladation mechanism for acyclic monoallylic diols, calculations and experiments strongly suggest that hydrogen bonding templates a hydroxyl group and Pd addition across the alkene and provides a low energy pathway via anti-addition (anti-oxypalladation) followed by intramolecular proton transfer and anti-elimination of water. This anti-addition, anti-elimination pathway also provides a simple rationale for the observed stereospecificity. For bicyclic monoallylic diol compounds, Pd(II) is capable of promoting either anti- or syn-addition. In addition, palladium chloride ligands can mediate proton transfer to promote dehydration when direct intramolecular proton transfer between diol groups is impossible.

H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters form a family of molecular motor proteins that couple ATP hydrolysis to substrate translocation across cell membranes. Each nucleotide binding domain of ABC-transporters contains a highly conserved H-loop histidine residue, whose precise mechanistic role in motor functions has remained elusive. By using combined quantum mechanical and molecular mechanical (QM/MM) calculations, we showed that the conserved H-loop residue H662 in E. coli HlyB, a bacterial ABC-transporter, can act first as a general acid and then as a general base to facilitate proton transfer in ATP hydrolysis. Without the assistance of H662, direct proton transfer from the lytic water to ATP results in a substantially higher barrier height. Our findings suggest that the essential function of the H-loop residue H662 is to provide a "chemical linchpin" that shuttles protons between reactants through a relay mechanism, thereby catalyzing ATP hydrolysis in HlyB.

Computer modeling on the tautomerization of sulbactam intermediate in SHV-1 β-lactamases: E166A mutant vs. wild type

We present a theoretical study for the tautomerization of sulbactam intermediates in different SHV-1 β-lactamases: E166A and wild-type (WT). Molecular dynamics (MD) simulations were employed and hydrogen bonds network around active site was found different between the WT and E166A acyl-enzymes. In E166A, Asn170 restricts the C5C6 bond rotation, thus stabilizes the dihedral angle N4C5C6C7 of imine to a trans conformation. The DFT calculations (B3LYP/6-31+G* and B3LYP/6-31++G**) were performed on tautomerization reactions. Two mechanisms including direct and stepwise proton transfer reactions were proposed based on the MD results. In E166A, the substrate carboxyl group acts as a relay station which assists the proton transfer with a very low energy barrier. However, in WT, such stepwise mechanism is difficult to proceed because of the large separation between C6 and substrate carboxyl group. Our results explain why E166A SHV-1 β-lactamases forms greater population of trans-enamine than WT.

Mechanistic Insights on Organocatalytic Enantioselective Decarboxylative Protonation by Epicinchona-Thiourea Hybrid Derivatives

Mechanism and the origin of enantioselectivity in the decarboxylative protonation of α-amino malonate hemiester promoted by epicinchona-thiourea hybrid organocatalyst is established by using the DFT(M06-2X/6-311+G**//ONIOM2) computational methods. The origin of stereoselectivity rendered by this hybrid bifunctional catalyst in asymmetric protonation is investigated for the first time using suitable transition-state models. A detailed conformational analysis of N-[3,5-bis(trifluoromethyl)]phenylthiourea-based epicinchonidine reveals the potential for a bifunctional mode of activation of the substrate α-amino malonate hemiester through hydrogen bonding. Six different conformer families differing in characteristic dihedral angles are identified within a range of 16 kcal/mol with respect to the lowest energy conformer. Different likely mechanistic pathways obtained through detailed analysis of the transition states and intermediates are compared. It is identified that in the preferred pathway, the decarboxylation is followed by a direct proton transfer from the chiral quinuclidinium moiety to the enolate carbon as opposed to a conventional protonation at the enolate oxygen followed by a keto-enol tautomerization. The factors responsible for high levels of observed stereoselectivity are traced to interesting hydrogen-bonding interactions offered by the thiourea-cinchona bifunctional framework. The predicted stereoselectivities using computed Gibbs free energies of diastereomeric transition states are in fair agreement with the experimental stereoselectivities.

Directionality of Dihydrogen Bonds: The Role of Transition Metal Atoms

A theoretical study on two series of electron-rich group 8 hydrides is carried out to evaluate involvement of the transition metal in dihydrogen bonding. To this end, the structural and electronic parameters are computed at the DFT/B3PW91 level for hydrogen-bonded adducts of [(PP(3))MH(2)] and [Cp*MH(dppe)] (M = Fe, Ru, Os; PP(3) = κ(4)-P(CH(2)CH(2)PPh(2))(3), dppe = κ(2)-Ph(2)PCH(2)CH(2)PPh(2)) with CF(3)CH(2)OH (TFE) as proton donor. The results are compared with those of adduct [Cp(2)NbH(3)]⋅TFE featuring a "pure" dihydrogen bond, and classical hydrogen bonds in pyridine⋅TFE and Me(3)N⋅TFE. Deviation of the H⋅⋅⋅H-A fragment from linearity is shown to originate from the metal participation in dihydrogen bonding. The latter is confirmed by the electronic parameters obtained by NBO and AIM analysis. Considered together, orbital interaction energies and hydrogen bond ellipticity are salient indicators of this effect and allow the MH⋅⋅⋅HA interaction to be described as a bifurcate hydrogen bond. The impact of the M⋅⋅⋅HA interaction is shown to increase on descending the group, and this explains the experimental trends in mechanisms of proton-transfer reactions via MH⋅⋅⋅HA intermediates. Strengthening of the M⋅⋅⋅H interaction in the case of electron-rich 5d metal hydrides leads to direct proton transfer to the metal atom.

Metal Hydride and Ligand Proton Transfer Mechanism for the Hydrogenation of Dimethyl Carbonate to Methanol Catalyzed by a Pincer Ruthenium Complex

The hydrogenation of dimethyl carbonate to methanol catalyzed by a PNN-ligated ruthenium complex (PNN)Ru(CO)(H) was studied computationally using the density functional theory at the range-separated and dispersion-corrected ωB97X-D functional level in conjunction with an all-electron 6-31++G(d,p) basis set (Stuttgart ECP28MWB basis set for Ru). A direct metal hydride and ligand proton transfer mechanism with three cascade catalytic cycles for the hydrogenation of dimethyl carbonate, methyl formate, and formaldehyde to methanol is proposed. The resting state in the catalytic reaction is the trans dihydride complex trans-(PNN)Ru(H)2(CO). Calculation results indicate that the rate-determining step in the whole reaction is the formation of the second methanol molecule through simultaneous breaking of a C–OCH3 bond and transferring a ligand methylene proton to the dissociated CH3O– in the catalytic cycle for hydrogenation of methyl formate. The essential role of the noninnocent PNN pincer ligand is to split H2 and assist methanol formation through the aromatization and dearomatization of the pyridine ring in the ligand. A new iron pincer complex, trans-(PNN)Fe(H)2(CO), is proposed and evaluated as a promising low-cost and high efficiency catalyst for this reaction.

Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium

A gene from the thermophilic archaeon Thermoplasma volcanium encoding an L-threonine dehydrogenase (L-ThrDH) with a predicted amino acid sequence that was remarkably similar to the sequence of UDP-galactose 4-epimerase (GalE) was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme was moderately thermostable, retaining more than 90% of its activity after incubation for 10 min at up to 70 °C. The catalytic residue was assessed using site-directed mutagenesis, and Tyr(137) was found to be essential for catalysis. To clarify the structural basis of the catalytic mechanism, four different crystal structures were determined using the molecular replacement method: L-ThrDH-NAD(+), L-ThrDH in complex with NAD(+) and pyruvate, Y137F mutant in complex with NAD(+) and L-threonine, and Y137F in complex with NAD(+) and L-3-hydroxynorvaline. Each monomer consisted of a Rossmann-fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed notable similarity to that of the GalE-like L-ThrDH from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via direct proton transfer driven by Tyr(137). The factors contributing to the thermostability of T. volcanium L-ThrDH were analyzed by comparing its structure to that of F. frigidimaris L-ThrDH. This comparison showed that the presence of extensive inter- and intrasubunit ion pair networks are likely responsible for the thermostability of T. volcanium L-ThrDH. This is the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like L-ThrDH.

“yl”-Oxygen Exchange in Uranyl(VI) Ion: A Mechanism Involving (UO2)2(μ-OH)22+ via U–Oyl–U Bridge Formation

Szabó and Grenthe (Inorg. Chem. 2007, 46, 9372-9378) suggested from NMR spectroscopy that the "yl"-oxygen exchange in dioxo uranium(VI) ion in acidic solution occurs via an OH-bridged binuclear complex (UO(2))(2)(μ-OH)(2)(2+). Here, an "yl"-oxygen exchange pathway involving the (UO(2))(2)(μ-OH)(2)(2+) is studied by B3LYP density functional theory calculations. The oxygen exchange takes place via an intramolecular proton shuttle between the oxygen atoms in (UO(2))(2)(μ-OH)(2)(H(2)O)(6)(2+). The direct proton transfer from the hydroxo bridge or from the coordinating water to the "yl"-oxygen in (UO(2))(2)(μ-OH)(2)(H(2)O)(6)(2+) appears to be negligible because of an exceedingly high activation barrier (∼170 kJ mol(-1)). The exchange mechanism in (UO(2))(2)(μ-OH)(2)(H(2)O)(6)(2+) can be described by a multistep pathway that leads to the formation of an oxo bridge between two uranyl(VI) centers (U-O(yl)-U bridge). The activation enthalpy Δ(‡)H of the reaction obtained at the B3LYP level is 94.7 kJ mol(-1) and is somewhat larger than the experimental value of 80 ± 14 kJ mol(-1). However, the discrepancy between theory and experiment is at the acceptable level. The formation of an oxo bridge between the two uranyl(VI) centers was found to be the key step in proton shuttling, indicating that uranyl(VI) complexes with a stable oxo bridge (such as trinuclear (UO(2))(3)(μ(3)-O)(OH)(3)(+)) may have even faster "yl"-oxygen exchange rates than (UO(2))(2)(μ-OH)(2)(2+).

The effect of hydrogen bonding on the excited-state proton transfer in 2-(2′-hydroxyphenyl)benzothiazole: a TDDFT molecular dynamics study

The dynamics of the excited-state proton transfer (ESPT) in a cluster of 2-(2'-hydroxyphenyl)benzothiazole (HBT) and hydrogen-bonded water molecules was investigated by means of quantum chemical simulations. Two different enol ground-state structures of HBT interacting with the water cluster were chosen as initial structures for the excited-state dynamics: (i) an intramolecular hydrogen-bonded structure of HBT and (ii) a cluster where the intramolecular hydrogen bond in HBT is broken by intermolecular interactions with water molecules. On-the-fly dynamics simulations using time-dependent density functional theory show that after photoexcitation to the S(1) state the ESPT pathway leading to the keto form strongly depends on the initial ground state structure of the HBT-water cluster. In the intramolecular hydrogen-bonded structures direct excited-state proton transfer is observed within 18 fs, which is a factor two faster than proton transfer in HBT computed for the gas phase. Intermolecular bonded HBT complexes show a complex pattern of excited-state proton transfer involving several distinct mechanisms. In the main process the tautomerization proceeds via a triple proton transfer through the water network with an average proton transfer time of approximately 120 fs. Due to the lack of the stabilizing hydrogen bond, intermolecular hydrogen-bonded structures have a significant degree of interring twisting already in the ground state. During the excited state dynamics, the twist tends to quickly increase indicating that internal conversion to the electronic ground state should take place at the sub-picosecond scale.

Excited-State Intermolecular Proton Transfer of Firefly Luciferin V. Direct Proton Transfer to Fluoride and Other Mild Bases

We studied the direct proton transfer (PT) from electronically excited D-luciferin to several mild bases. The fluorescence up-conversion technique is used to measure the rise and decay of the fluorescence signals of the protonated and deprotonated species of D-luciferin. From a base concentration of 0.25 M or higher the proton transfer rates to the fluoride, dihdyrogen phosphate or acetate bases are fast and comparable. The fluorescence signals are nonexponential and complex. We suggest that the fastest decay component arises from a direct proton transfer process from the hydroxyl group of D-luciferin to the mild base. The proton donor and acceptor molecules form an ion pair prior to photoexcitation. Upon photoexcitation solvent rearrangement occurs on a 1 ps time-scale. The PT reaction time constant is ∼2 ps for all three bases. A second decay component of about 10 ps is attributed to the proton transfer in a contact pair bridged by one water molecule. The longest decay component is due to both the excited-state proton transfer (ESPT) to the solvent and the diffusion-assisted PT process between a photoacid and a base pair positioned remotely from each other prior to photoexcitation.

Structural Photodynamics of Camptothecin, an Anticancer Drug in Aqueous Solutions

Steady-state and time-resolved picosecond emission studies were carried out to study the role of the proton concentration in the acid-base properties of the anticancer drug camptothecin (CPT) in its ground and electronically first excited states. The results show that, under acidic conditions, the excited-state proton-transfer (ESPT) reaction is irreversible, in contrast to previous literature data. We found that the prototropic species are equilibrated at the excited state (pK(a)* = 1.85) only in a restricted range of pH (1.5 < pH < 3), whereas only one species, either the neutral form (τ(N) = 3.76 ns) or the protonated form (τ(C) = 2.83 ns), can be detected at pH > 3 and pH < 1.5, respectively. The proton motion from the acidic solution to the neutral form in the pH 1-2 domain is diffusion-controlled. Within the range of pH 1-2, the reaction rate constant for the formation (k(d)) of the encounter complex between the proton and the neutral form ranges from 1.17 × 10(10) to 7.33 × 10(10) M(-1) s(-1), respectively. Under more acidic conditions (pH 0.9-0.95), the protonation of CPT does not depend on the diffusive step, because of the large amount of protons. The direct proton-transfer rate constant (k(DPT)*) increases with the proton concentration (time constants change from 24 ps to ∼1 ns at pH 0.9 and 2, respectively). The number of protons involved in the proton transfer changes from approximately one, for the diffusive regime, to approximately four, for the static regime. We found good agreement between the Birks model for equilibrated flourophores and the Debye-Smoluchowski equation (DSE) to accurately explain the ESPT reaction of CPT with acidic water in the reversible range. The proton motion at pH 2 (equilibrium range) exhibits diffusion-controlled behavior and can be explained using the Smoluchowski model. Our results show that the interaction of CPT with acidic water depends on the concentration of the acid, which changes the nature of both the structure and dynamics.

Interaction and Inhibitory Effect of Ammonium Cation in the Oxygen Evolving Center of Photosytem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.

AB INITIOCOMPUTATIONAL INVESTIGATIONS ON THE GAS-PHASE HOMODIMERIZATION AND KETO-ENOL TAUTOMERISM OF THE MONOCHALCOGENOCARBOXYLIC ACIDSCH3C(=O)XH (X = S, Se, Te)

Computational investigations on the gas-phase homodimerization and the keto-to-enol tautomerism of monochalcogenocarboxylic acids, CH3C(=O)XH (X = S, Se, Te) , were performed using ab initio molecular orbital methods (HF and MP2) with the 6-311+G(d, p) and 6-311+G(2df, 2df) basis sets. Calculated results indicate that the dimerization enthalpy values, ΔH, for the enol-dimers, [CH3C(=X)OH]2(X = S, Se, Te) , are notably higher than those for the corresponding keto-dimers, [CH3C(=O)XH]2(X = S, Se, Te) , while the ΔH values decrease as the electronegativity of chalcogen atom is lowered, for both the keto- and enol-dimers. It is found that the homodimerization of monochalcogenocarboxylic acids is thermodynamically unfavorable because the releasing dimerization heat cannot overcome the loss of entropy. This is contrasted with the case in the carboxylic acid, where the much higher dimerization enthalpy is responsible for the favorable dimerization in the gas phase. Our results also suggest that the tautomeric reactions of the monochalcogenocarboxylic acids in the gas phase may proceed by an eight-membered ring TS with intermolecular double proton transfer and lower tautomeric barrier, instead of by direct intramolecular proton transfer in monomer with a highly strained four-membered TS. The geometrical and energetic characteristics of the homodimers and tautomeric TSs are also further elucidated by NBO analysis.

Electrophilic Addition Reaction of Ethene with Hydrogen Chloride on Cold Molecular Films: Evidence of Ethyl Cationic Intermediate

We studied the initial-stage mechanism of the electrophilic addition reaction of ethene with HCl by examining the interactions between ethene and HCl on water-ice and frozen molecular films at temperatures of 80-140 K. Cs(+) reactive ion scattering (RIS) and low-energy sputtering (LES) techniques were used to probe the reaction intermediates that were kinetically trapped on the surface, in conjunction with temperature-programmed desorption (TPD) mass spectrometry to monitor the desorbing species. The reaction initially produced the π complex of HCl and ethene at temperatures below about 93 K and an "ethyl cationic species" at temperatures below about 100 K. The ethyl cationic species was formed via direct proton transfer from the HCl molecule to ethene with the assistance of water solvation, rather than via the interaction of hydronium ions and ethene. At high temperatures, this species dissociated into ethene and hydronium and chloride ions. The reaction did not, however, complete the final transition state on the ice surface to produce ethyl chloride. The observation gives evidence that the electrophilic addition reaction of ethene occurs through an ethyl-like intermediate with an ionic character.

Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily

The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.

The role of water in the proton transfer reaction mechanism in tryptophan

The role of water in the proton transfer mechanism between the carboxylic and the amino group in tryptophan was studied through the direct, intramolecular reaction in presence of a continuum and the single water molecule mediated, intermolecular reaction in vacuum and in presence of a continuum. The introduction of the continuum reduced the activation barriers for the proton transfer from the neutral form and stabilized the zwitterion in both cases. The reaction force and the reaction electronic flux along the intrinsic reaction coordinate allowed in combination with a Natural Bond Order Analysis a more detailed description of the influence of water on the reaction mechanism. Represented as a continuum model, water reduced the energy required for bond reorganization and did not alter the first part of the intermolecular reaction, characterized through the approximation of the water molecule to the functional groups of the amino acid and a polarization of the system. The absence of a water molecule in the intramolecular proton transfer with continuum changed the reaction coordinate to a reduction of the angle between the functional groups of the amino acid leaving the energy required for bond formation unaffected. Thus, the smaller activation barrier obtained in the direct intramolecular proton transfer with continuum model in comparison to the water mediated reaction, originates from the energetically more favorable angle reduction in comparison the approximation of a water molecule.