A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

Rapid photodynamics of vitamin B6 coenzyme pyridoxal 5'-phosphate and its Schiff bases in solution

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is an important cofactor for numerous enzymes in amine and amino acid metabolism. Presented here is the first femtosecond transient absorption study of free PLP and two Schiff bases, PLP-valine and PLP-alpha-aminoisobutyric acid (AIB), in solution. Photoexcitation of free PLP leads to efficient triplet formation with an internal conversion rate that increases with increasing pH. The measured excited-state kinetics of the PLP-valine Schiff base exhibits a dramatic deuterium dependence as a result of excited-state proton transfer (ESPT) of the Calpha hydrogen in the amino acid substrate. This is consistent with formation of the key reaction carbanionic intermediate (quinonoid), which is resonance stabilized by the electron-deficient, conjugated pi system of the Schiff base/pyridine ring. The transient absorption signals of the PLP-Schiff base with alpha-methylalanine (2-aminoisobutyric acid), which does not have a Calpha proton, lack an observable deuterium effect, verifying ESPT formation of the quinonoid intermediate. In contrast to previous studies, no dependence on the excitation wavelength of the femtosecond kinetics is observed with PLP or PLP-valine, which suggests that a rapid (<250 fs) tautomerization occurs between the enolimine (absorbing at 330 nm) and ketoenamine (absorbing at 410 nm) tautomers in solution.     

A density functional theory study on mechanisms of [4 + 2] annulation of enal with α-methylene cycloalkanone catalyzed by N-heterocyclic carbene

Density functional theory was used to study the reaction mechanisms of the N-heterocyclic carbene (NHC)-catalyzed [4 + 2] annulation reaction between enal and α-methylene cycloalkanone for the formation of tricyclic benzopyran-2-one. The simulations suggest that the energy-favorable catalytic cycle includes five steps: (a) the nucleophilic addition of enal by NHC catalyst; (b) [1, 2]-proton transfer for the formation of Breslow intermediate; (c) [1, 4]-proton transfer for the formation of enolate intermediate; (d) the [4 + 2] cycloaddition process, product formation; and (e) catalyst regeneration. The proton transfer process was particularly designed in two independent ways, the direct proton transfer and the Brønsted acid DBU∙H⁺-mediated proton transfer. Our study reveals that [1, 2]-proton transfer process is the rate-determining step with the accessible energy barrier, which agrees with the experimental observation. Further analysis of the global reaction index confirmed that NHC was primary used as a Lewis base during the reaction processes. The frontier molecular orbital (FMO) analysis indicates that the introduction of the NHC catalyst facilitates the reaction to occur due to the narrower energy gap of FMO.     

The role of Acid catalysis in the baeyer-villiger reaction. A theoretical study

Quantum mechanical calculations at the B3LYP/6-311+G(d,p) level have examined the overall mechanism of the Baeyer-Villiger (BV) reaction with peroxyacetic acid. A series of reactions that include both the addition step and the subsequent alkyl group migration step included ketones, acetone, t-butyl methyl ketone, acetophenone, cyclohexyl methyl ketone, and cyclohexyl phenyl ketone. The combined data suggested that the first step for addition of the peroxyacetic acid oxidation catalyst to the ketone carbonyl to produce the Criegee or tetrahedral intermediate is rate-limiting and has activation barriers that range from 38 to 41 kcal/mol without the aid of a catalyst. The rate of addition is markedly reduced by the catalytic action of a COOH functionality acting as a donor-acceptor group affecting both its proton transfer to the ketone C═O oxygen in concert with transfer of the OOH proton to the carboxylic acid carbonyl. The second or alkyl group migration step has a much reduced activation barrier, and its rate is not markedly influenced by acid catalysis. The rate of both steps in the BV reaction is greatly influenced by the catalytic action of very strong acids.     

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.     

New approach to biomimetic transamination using bifunctional [1,3]-proton transfer catalysis in thioxanthenyl dioxide imines

A pyridoxamine equivalent, 9-aminothioxanthene 10,10-dioxide, has been designed that is capable of affording transamination in good to excellent yields of natural as well as artificial amino acids. Amidines and guanidines in catalytic amounts were capable of performing [1,3]-proton transfer in the imines under mild conditions, whereas various simple amines failed. The use of chiral catalysts resulted in modest asymmetric induction (ee < or = 45%). The electronic dependence in para-substituted phenyl glyoxylate imines, isotope effects, and computational studies support a stepwise, bifunctional mechanism for amidine and guanidine catalysts. Attempts toward an autocatalytic model system are described.     

Kinetic studies on the thermal cis-trans isomerization of 1, 3-diphenyltriazene in aqueous solution. Effects Of acids and bases

The thermal cis-to-trans isomerization of 1,3-diphenyltriazene (DPT) has been investigated in buffered aqueous solutions by means of laser-flash photolysis techniques. The cis-to-trans isomerization process is found to be catalyzed by general acids and general bases as a result of acid/base-promoted 1,3-prototropic rearrangements. Acid catalysis is attributed to rate-limiting proton transfer to the nitrogen-nitrogen double bond of cis-DPT, whereas base catalysis is attributed to rate-limiting base-promoted ionization of the amino nitrogen of cis-DPT leading to the isomerization. In addition, a process ascribed to the interconversion of cis rotamers through hindered rotation around the nitrogen-nitrogen single bond is also observed; at high pH this process becomes rate-limiting.     

Elucidation of the reaction mechanisms and diastereoselectivities of phosphine-catalyzed [4 + 2] annulations between allenoates and ketones or aldimines

The phosphine-catalyzed [4 + 2] annulations between allenoates and electron-poor trifluoromethyl ketones or N-tosylbenzaldimine dipolarophiles have been investigated in continuum solvation using density functional theory (DFT) calculations. The detailed reaction mechanisms as well as the high cis-diastereoselectivities of the reactions have been firstly clarified. Our calculated results reveal that the whole catalytic process is presumably initiated with the nucleophilic attack of phosphine catalyst at the allenoate to produce the zwitterionic intermediate , which subsequently undergoes γ-addition to the electron-poor C[double bond, length as m-dash]O (or C[double bond, length as m-dash]N) dipolarophile to form another intermediate . The following [1,3] hydrogen shift of is demonstrated to proceed via two consecutive proton transfer steps without the assistance of protic solvent: the anionic O6 (or N6) of first acts as a base catalyst to abstract a proton from C1 to produce the intermediate , and then the OH (or NH) group can donate the acidic proton to C3 to complete the [1,3] hydrogen shift and generate the intermediate . Finally, the intramolecular Michael-type addition followed by the elimination of catalyst furnishes the final product. High cis-diastereoselectivities are also predicted for both the two reactions, which is in good agreement with the experimental observations. For the reaction of allenoates with trifluoromethyl ketones, the first proton transfer is found to be the diastereoselectivity-determining step. The cumulative effects of the steric repulsion, electrostatic interaction as well as other weak interactions appear to contribute to the relative energies of transition states leading to the diastereomeric products. On the contrary, in the case of N-tosylbenzaldimines, the Michael-type addition is found to be the diastereoselectivity-determining step. Similarly, steric repulsion, as well as electrostatic interaction is also identified to be the dominant factors in controlling the high cis-diastereoselectivity of this reaction.     

Pyridoxal-5'-phosphate-dependent catalytic antibodies

Cofactors--i.e., metal ions and coenzymes--extend the catalytic scope of enzymes and might have been among the first biological catalysts. They may be expected to efficiently extend the catalytic potential of antibodies. Monoclonal antibodies (MAbs) against Nalpha-phosphopyridoxyl-L-lysine were screened for 1) binding of 5'-phosphopyridoxyl amino acids, 2) binding of the planar Schiff base of pyridoxal-5'-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3) catalysis of the PLP-dependent alpha, beta-elimination reaction with beta-chloro-D/L-alanine. Antibody 15A9 fulfilled all criteria and was also found to catalyze the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k'cat = 0.42 min(-1) with D-alanine at 25 degrees C). No other reactions with either D- or L-amino acids were detected. PLP markedly contributes to catalytic efficacy-it is a 10(4) times more efficient acceptor of the amino group than pyruvate. The antibody ensures reaction specificity, stereospecificity, and substrate specificity, and further accelerates the transamination reaction (k'cat(Ab)/k'cat(PLP) = 5 x 10(3)). The successive screening steps simulate the selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.     

DFT study of the mechanisms of in water Au(I)-catalyzed tandem [3,3]-rearrangement/Nazarov reaction/[1,2]-hydrogen shift of enynyl acetates: a proton-transport catalysis strategy in the water-catalyzed [1,2]-hydrogen shift

A computational study with the Becke3LYP density functional was carried out to elucidate the mechanisms of Au(I)-catalyzed reactions of enynyl acetates involving tandem [3,3]-rearrangement, Nazarov reaction, and [1,2]-hydrogen shift. Calculations indicate that the [3,3]-rearrangement is a two-step process with activation free energies below 10 kcal/mol for both steps. The following Nazarov-type 4pi electrocyclic ring-closure reaction of a Au-containing dienyl cation is also easy with an activation free energy of 3.2 kcal/mol in CH2Cl2. The final step in the catalytic cycle is a [1,2]-hydride shift, and this step is the rate-limiting step (with a computed activation free energy of 20.2 kcal/mol) when dry CH2Cl2 is used as the solvent. When this tandem reaction was conducted in wet CH2Cl2, the [1,2]-hydride shift step in dry solution turned to a very efficient water-catalyzed [1,2]-hydrogen shift mechanism with an activation free energy of 16.4 kcal/mol. Because of this, the tandem reaction of enynyl acetates was found to be faster in wet CH2Cl2 as compared to the reaction in dry CH2Cl2. Calculations show that a water-catalyzed [1,2]-hydrogen shift adopts a proton-transport catalysis strategy, in which the acetoxy group in the substrate is critical because it acts as either a proton acceptor when one water molecule is involved in catalysis or a proton-relay stabilizer when a water cluster is involved in catalysis. Water is found to act as a proton shuttle in the proton-transport catalysis strategy. Theoretical discovery of the role of the acetoxy group in the water-catalyzed [1,2]-hydrogen shift process suggests that a transition metal-catalyzed reaction involving a similar hydrogen shift step can be accelerated in water or on water with only a marginal effect, unless a proton-accepting group such as an acetoxy group, which can form a hydrogen bond network with water, is present around this reaction's active site.     

A New Pyridoxal Derivative for Transamination of N‐Terminus of Proteins

A new pyridoxal‐5‐phosphate (PLP) derivative FHMDP was developed for the transamination of different peptides with three most hindered amino acid residues (Leu, Ile, Val) as their N‐terminus. Compared to the previously reported reactions of PLP derivatives, the N‐terminus transamination could be accomplished efficiently with the new compound.     

Catalytic mechanism of 6-phosphogluconate dehydrogenase: a theoretical investigation

Density functional calculations are employed to theoretically explore the mechanism of all elementary reaction steps involved in the catalytic reaction of 6-phosphogluconate dehydrogenase (6PGDH). The model systems we choose for the enzyme contain the essential parts of the cofactor (NADP+), the substrate 6-phosphogluconate (6PG), and some key residues (Lys183 and Glu190) in the active site of sheep liver 6PGDH. The effect of the apoenzyme electrostatic environment on the studied reaction is treated by the self-consistent reaction-field method. Our calculations demonstrate that the first step of the catalytic reaction is the formation of a 3-keto 6PG intermediate, which proceeds through a concerted transition state involving a hydride transfer from 6PG to NADP+, and a proton transfer from 6PG to Lys183. The second step is the elimination of a CO2 molecule from 6-PG, concomitant with a proton transfer from Lys183 to 6-PG. In the final step, a concerted double proton transfer (one from Glu190 to the substrate, another from the substrate to Lys183) results in the final product, the keto form of ribulose 5-phosphate (Ru5P). The rate-limiting step is the formation of a 3-keto 6PG intermediate, with a free energy barrier of 22.7 kcal/mol at room temperature in the protein environment, and all three steps are calculated to be thermodynamically favorable. These results are in good agreement with the general acid/general base mechanism suggested from previous experiments for the 6PGDH reaction.     

On the role of the conserved aspartate in the hydrolysis of the phosphocysteine intermediate of the low molecular weight tyrosine phosphatase

The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine phosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, general base-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked. However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do not reveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of water is difficult to reconcile with the known gas-phase proton affinities and solution phase pK(a)'s of aspartic acid and water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. To understand the hydrolysis mechanism, we have used high-level density functional methods of quantum chemistry combined with continuum electrostatics models of the protein and the solvent. Our calculations do not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygen directly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transition state, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to proton transfer to the base is greatly diminished; the aspartate can abstract a proton only after the transition state, a result consistent with experimental solvent isotope effects for this enzyme and with established precedents for phosphomonoester hydrolysis.     

Reaction of imidazole with toluene-4-sulfonate salts of substituted phenyl N-methylpyridinium-4-carboxylate esters: special base catalysis by imidazole

The reaction of imidazole in aqueous solution with toluene-4-sulfonate salts of substituted phenyl N-methylpyridinium-4-carboxylate esters obeys the rate law: k(obs) - k(background) = k2[Im] + k3[Im]2 where [Im] is the imidazole concentration present as free base. The parameters k2 and k3 fit Br?nsted type free energy correlations against the pKa of the leaving phenol with betaLg values of -0.65 and -0.42 respectively. The imidazolysis is insensitive to catalysis by general bases and yet k3 for the 3-cyanophenyl ester possesses a deuterium oxide solvent isotope effect of 4.43 consistent with rate limiting proton transfer. A special catalytic function is proposed for decomposition of the tetrahedral addition intermediate (T+/-) via k3 whereby the catalytic imidazole interacts electrophilically with the leaving phenolate ion and removes a proton from the nitrogen in the rate limiting step with subsequent non-rate limiting ArO-C bond fission. This is consistent with the change in effective charge on the leaving oxygen in the transition structure of k3 which is more positive (-0.42) than that expected (-0.60) for the equilibrium formation of the zwitterion intermediate. The catalytic function at the leaving oxygen is likely to be an electrophilic role of the NH as a hydrogen bond donor. In the k2 step the deuterium oxide solvent isotope effect of 1.51 for the 3-cyanophenyl ester and the betaLg of -0.65 are consistent with rate limiting expulsion of the phenolate ion from the T+/- intermediate. The absence of general base catalysis of imidazolysis rules out the established mechanism for aminolysis of esters where T+/- is stabilised by a standard rate limiting proton transfer. The kinetically equivalent term for k3 where T- reacts with the imidazolium ion as an acid catalyst would require this step to be rate limiting and involve proton transfer not consistent with departure of the good aryl oxide leaving group.     

Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD)

Extensive combined quantum mechanical (B3LYP/6‐31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton‐transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2‐pyrimidinone. The rate‐determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.     

Synthesis of a potential bifunctional mimic of transaminases.

As a potential bifunctional mimic of transaminases 3,7-dimethyl-10-[3-(4- aminomethyl-5-hydroxy-6-methyl-3-pyridyl)propyl]-3,7,10- triazatricyclo[3.3.3.0(1,5)] undecane (I) has been synthesized by attaching 3,7-dimethyl-3,7,10-triazatricyclo [3.3.3.0(1,5)]undecane (II) to a pyridoxamine nucleus via an all-carbon chain. The chain length between the pyridine ring and II is restricted to three atom units so that the possibility for II to act bifunctionally during the transamination is maximized. In its protonated form, the nitrogen closest to the pyridine ring cannot deliver the proton intramolecularly to the alpha-carbon of the developing amino acid. To make the synthetic route generally applicable, introduction of the side-arm base is arranged at a later stage of the synthesis so that different di- or poly-amines can easily be used in place of II to prepare other target molecules that might possess bifunctional catalytic activity. This arrangement also greatly reduces the polarity and water-solubility of the intermediates and the purification of these compounds thus becomes much easier. The method of introducing the amino functionality at the C-4 methylene group described herein provides an alternative to that currently in use (reduction of oximes).     

The reaction mechanism of the hydroamination of alkenes catalyzed by gold(I)-phosphine: the role of the counterion and the N-nucleophile substituents in the proton-transfer step

The reaction mechanism of the gold(I)-phosphine-catalyzed hydroamination of 1,3-dienes was analyzed by means of density functional methods combined with polarizable continuum models. Several mechanistic pathways for the reaction were considered and evaluated. It was found that the most favorable series of reaction steps include the ligand substitution reaction in the catalytically active Ph3PAuOTf species between the triflate and the substrate, subsequent nucleophile attack of the N-nucleophile (benzyl carbamate) on the activated double bond, which is followed by proton transfer from the NH2 group to the unsaturated carbon atom. The latter step, the most striking one, was analyzed in detail, and a novel pathway involving tautomerization of benzyl carbamate nucleophile assisted by triflate anion acting as a proton shuttle was characterized by the lowest barrier, which is consistent with experimental findings.     

Brønsted Acid Catalyzed Morita–Baylis–Hillman Reaction: A New Mechanistic View for Thioureas Revealed by ESI‐MS(/MS) Monitoring and DFT Calculations

A Morita–Baylis–Hillman (MBH) reaction catalyzed by thiourea was monitored by ESI‐MS(/MS) and key intermediates were intercepted and characterized. These intermediates suggest that thiourea acts as an organocatalyst in all steps of the MBH reaction cycle, including the rate‐limiting proton‐transfer step. DFT calculations, performed for a model MBH reaction between formaldehyde and acrolein with trimethylamine as base and in the presence or the absence of thiourea, suggest that thiourea accelerates MBH reactions by decreasing the transition‐state (TS) energies through bidentate hydrogen bonding throughout the whole catalytic cycle. In the rate‐limiting proton‐transfer step, the thiourea acts not as a proton shuttle, but as a Brønsted acid stabilizing the basic oxygen center that is formed in the TS.     

Concerted or stepwise hydrogen transfer in the transfer hydrogenation of acetophenone catalyzed by ruthenium-acetamido complex: a theoretical mechanistic investigation

In this paper, the mechanism of transfer hydrogenation of acetophenone catalyzed by ruthenium-acetamido complex was studied using density function theory (DFT) method. The catalytic cycle of transfer hydrogenation consists of hydrogen transfer (HT) step and dehydrogenation (DH) step of isopropanol (IPA). Inner sphere mechanism (paths 1 and 7) and outer sphere mechanism (paths 2-6) in HT step are fully investigated. Calculated results indicate that DH step of IPA (from (i)1 to (i)2) is the rate-determining step in the whole catalytic cycle, which has a potential energy barrier of 16.2 kcal/mol. On the other hand, the maximum potential energy barriers of paths 1-7 in the HT step are 5.9, 12.7, 24.4, 16.8, 23.7, 7.2, and 6.1 kcal/mol, respectively. The inner sphere pathways (paths 1 and 7) are favorable hydrogen transfer modes compared with outer sphere pathways, and the proton transferred to the oxygen atom of acetophenone comes from the hydroxyl group but not from amino group of acetamido ligand. Those theoretical results are in agreement with experimental report. However, in view of this DFT study in the inner sphere mechanism of HT step, hydride transfer and proton transfer are concerted and asynchronous hydrogen transfer but not a stepwise one, and hydride transfer precedes proton transfer in this case.