Protonation‐Dependent Base Flipping at Neutral pH in the Catalytic Triad of a Self‐Splicing Bacterial Group II Intron

NMR spectroscopy has revealed pH‐dependent structural changes in the highly conserved catalytic domain 5 of a bacterial group II intron. Two adenines with pK_a values close to neutral pH were identified in the catalytic triad and the bulge. Protonation of the adenine opposite to the catalytic triad is stabilized within a G(syn)–AH⁺(anti) base pair. The pH‐dependent anti‐to‐syn flipping of this G in the catalytic triad modulates the known interaction with the linker region between domains 2 and 3 (J23) and simultaneously the binding of the catalytic Mg²⁺ ion to its backbone. Hence, this here identified shifted pK_a value controls the conformational change between the two steps of splicing.     

Role of Asp102 in the catalytic relay system of serine proteases: a theoretical study

The role of Asp102 in the catalytic relay system of serine proteases is studied theoretically by calculating the free energy profiles of the single proton-transfer reaction by the Asn102 mutant trypsin and the concerted double proton-transfer reaction (so-called the charge-relay mechanism) of the wild-type trypsin. For each reaction, the reaction free energy profile of the rate-determining step (the tetrahedral intermediate formation step) is calculated by using ab initio QM/MM electronic structure calculations combined with molecular dynamics-free energy perturbation method. In the mutant reaction, the free energy monotonically increases along the reaction path. The rate-determining step of the mutant reaction is the formation of tetrahedral intermediate complex, not the base (His57) abstraction of the proton from Ser195. In contrast to the single proton-transfer reaction of the wild-type, MD simulations of the enzyme-substrate complex show that the catalytically favorable alignment of the relay system (the hydrogen bonding network between the mutant triad, His57, Asn102, and Ser195) is rarely observed even in the presence of a substrate at the active site. In the double proton-transfer reaction, the energy barrier is observed at the proton abstraction step, which corresponds to the rate-determining step of the single proton-transfer reaction of the wild-type. Although both reaction profiles show an increase of the activation barrier by several kcals/mol, these increases have different energetic origins: a large energetic loss of the electrostatic stabilization between His57 and Asn102 in the mutant reaction, while the lack of stabilization by the protein environment in the double proton-transfer reaction. Comparing the present results with the single proton transfer of the wild-type, Asp102 is proven to play two important roles in the catalytic process. One is to stabilize the protonated His57, or ionic intermediate, formed during the acylation, and the other is to fix the configuration around the active site, which is favorable to promote the catalytic process. These two factors are closely related to each other and are indispensable for the efficient catalysis. Also the present calculations suggest the importance of the remote site interaction between His57 and Val213-Ser214 at the catalytic transition state.     

Direct ab‐initio molecular dynamics study on the radiation effects on catalytic triad composed of Ser‐His‐Glu residues

High energy irradiation to the hydrogen bonded system is important in relevance with the initial process of DNA and enzyme damages. In the present study, the effects of radiation to catalytic triad have been investigated by means of direct ab‐initio molecular dynamics (AIMD) calculation. As a model of the catalytic triad, Ser‐His‐Glu residue, which is one of the important enzymes in the acylation reaction, was examined. The ionization and electron attachment processes in Ser‐His‐Glu were investigated as the radiation effects. The direct AIMD calculation showed that a proton of His is spontaneously transferred to carbonyl oxygen of Glu after the ionization. However, the whole structure of catalytic triad was essentially kept after the ionization. On the other hand, in the case of the electron capture in the model catalytic triad Ser‐His‐Glu, the dissociation of Glu residue from [Ser‐His]^? was found as a product channel. The mechanism of ionization and electron capture process in the catalytic triad was discussed on the basis of theoretical results. © 2015 Wiley Periodicals, Inc.     

Theoretical perspectives on the reaction mechanism of serine proteases: the reaction free energy profiles of the acylation process

The reaction mechanism of serine proteases (trypsin), which catalyze peptide hydrolysis, is studied theoretically by ab initio QM/MM electronic structure calculations combined with Molecular Dynamics-Free Energy Perturbation calculations. We have calculated the entire reaction free energy profiles of the first reaction step of this enzyme (acylation process). The present calculations show that the rate-determining step of the acylation is the formation of the tetrahedral intermediate, and the breakdown of this intermediate has a small energy barrier. The calculated activation free energy for the acylation is approximately 17.8 kcal/mol at QM/MM MP2/(aug)-cc-pVDZ//HF/6-31(+)G/AMBER level, and this reaction is an exothermic process. MD simulations of the enzyme-substrate (ES) complex and the free enzyme in aqueous phase show that the substrate binding induces slight conformational changes around the active site, which favor the alignment of the reactive fragments (His57, Asp102, and Ser195) together in a reactive orientation. It is also shown that the proton transfer from Ser195 to His57 and the nucleophilic attack of Ser195 to the carbonyl carbon of the scissile bond of the substrate occur in a concerted manner. In this reaction, protein environment plays a crucial role to lowering the activation free energy by stabilizing the tetrahedral intermediate compared to the ES complex. The polarization energy calculations show that the enzyme active site is in a very polar environment because of the polar main chain contributions of protein. Also, the ground-state destabilization effect (steric strain) is not a major catalytic factor. The most important catalytic factor of stabilizing the tetrahedral intermediate is the electrostatic interaction between the active site and particular regions of protein: the main chain NH groups in Gly193 and Ser195 (so-called oxyanion hole region) stabilize negative charge generated on the carbonyl oxygen of the scissile bond, and the main chain carbonyl groups in Ile212 approximately Ser214 stabilize a positive charge generated on the imidazole ring of His57.     

The Role of Hydrogen Bond in Catalytic Triad of Serine Proteases

In order to investigate the origin of catalytic power for serine proteases, the role of the hydrogen bond in the catalytic triad was studied in the proteolysis process of the peptides chymotrypsin inhibitor 2 (CI2), MCTI-A, and a hexapeptide (SUB), respectively. We first calculated the free energy profile of the proton transfer between His and Asp residues of the catalytic triad in the enzyme-substrate state and transition state by employing QM/MM molecular dynamics simulations. The results show that a low-barrier hydrogen bond (LBHB) only forms in the transition state of the acylation of CI2, while it is a normal hydrogen bond in the acylation of MCTI-A or SUB. In addition, the change of the hydrogen bond strength is much larger in CI2 and SUB systems than in MCTI-A system, which decreases the acylation energy barrier significantly for CI2 and SUB. Clearly, a LBHB formed in the transition state region helps accelerate the acylation reaction. But to our surprise, a normal hydrogen bond can also help to decrease the energy barrier. The key to reducing the reaction barrier is the increment of hydrogen bond strength in the transition state state, whether it is a LBHB or not. Our studies cast new light on the role of the hydrogen bond in the catalytic triad, and help to understand the catalytic triad of serine proteases.     

A model study of the efficiency of the Asp–His–Ser triad

The observation of the Asp–His–Ser triad (Asp: aspartate, His: histidine, Ser: serine) triad both in mammalian and bacterial proteases suggests a special efficiency. A series of B3LYP/D95*(d,p) calculations on various [X–H^βY]^? dyads (as part of the [X–H^βY–H^αAc]^? model triad, HAc: acetic acid) made from eight different anions X^? and 15 different coupling elements H^βY was done to analyze the molecular origin of this efficiency. The X^? anion acts merely as an electron density donor independent of its chemical nature, and the evolutionary selection of Asp for the catalytic triad therefore seems to be caused by the pH of the triads environment. As the linking proton H^β moves from Y^? to X^?, electron density is effectively moved from X^? to Y^? thereby increasing the proton affinity (PA) of the [X–HY]^? dyad, which finally leads to the deprotonization of the HAc molecule. The degree to which the position of H^α controls the PA is dominatly determined by the coupling element HY. The model calculations indicate that 4‐methyl‐1H‐imidazole (HMim) is a very efficient coupling element, which suggest that the evolutionary convergence to the Asp–His–Ser is not only controlled by the ready availability of the imidazole motive in His but also by its high efficiency. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Acidity Constant (pKa) Calculation of Large Solvated Dye Molecules: Evaluation of Two Advanced Molecular Dynamics Methods

pH‐Sensitive dyes are increasingly applied on polymer substrates for the creation of novel sensor materials. Recently, these dye molecules were modified to form a covalent bond with the polymer host. This had a large influence on the pH‐sensitive properties, in particular on the acidity constant (pK_a). Obtaining molecular control over the factors that influence the pK_a value is mandatory for the future intelligent design of sensor materials. Herein, we show that advanced molecular dynamics (MD) methods have reached the level at which the pK_a values of large solvated dye molecules can be predicted with high accuracy. Two MD methods were used in this work: steered or restrained MD and the insertion/deletion scheme. Both were first calibrated on a set of phenol derivatives and afterwards applied to the dye molecule bromothymol blue. Excellent agreement with experimental values was obtained, which opens perspectives for using these methods for designing dye molecules.     

Structure–Reactivity Relationship in the Frustrated Lewis Pair (FLP)‐Catalyzed Hydrogenation of Imines

The autoinduced, frustrated Lewis pair (FLP)‐catalyzed hydrogenation of 16‐benzene‐ring substituted N‐benzylidene‐tert‐butylamines with B(2,6‐F₂C₆H₃)₃ and molecular hydrogen was investigated by kinetic analysis. The pK_a values for imines and for the corresponding amines were determined by quantum‐mechanical methods and provided a direct proportional relationship. The correlation of the two rate constants k₁ (simple catalytic cycle) and k₂ (autoinduced catalytic cycle) with pK_a difference between imine and amine pairs (ΔpK_a) or Hammett's σ parameter served as useful parameters to establish a structure–reactivity relationship for the FLP‐catalyzed hydrogenation of imines.     

Poly(L‐lysine)‐Induced Aggregation of Single‐Strand Oligo‐DNA‐Modified Gold Nanoparticles

Single‐strand oligo‐DNA‐modified Au nanoparticles (AuNPs) undergo aggregation in the presence of poly(L ‐lysine) (PLL), which is attributed to the interactions between the oligo‐DNA and PLL. These interactions between the oligo‐DNA and PLL were identified to be electrostatic when the lysine residues of PLL were positively charged and to be hydrogen bonding when the residues were deprotonated. The aggregation was promoted with an increase in the pH value at a pH level lower than the pK_a value of PLL (pK_a≈10.0) due to the gradual deprotonation of the lysine residues and thus suppressed electrostatic interactions between the positively charged lysine residues of PLL and the negatively charged backbone phosphate groups of the oligo‐DNA. At pH levels higher than the pK_a value of PLL, the aggregation was identified to be dominated by the hydrogen bonds between the bases of the oligo‐DNA and the deprotonated lysine residues of PLL. This study prompts the possibility that the spectral, and thus color, change of AuNPs upon aggregation can be used as a probe to follow the interactions between oligo‐DNA and polypeptides.     

Nucleophilic substitution reactions of diethyl 4‐nitrophenyl phosphate triester: Kinetics and mechanism

The reactions of diethyl 4‐nitrophenyl phosphate ( 1 ) with a series of nucleophiles: phenoxides, secondary alicyclic (SA) amines, and pyridines are subjected to a kinetic study. Under excess of nucleophile, all the reactions obey pseudo‐first‐order kinetics and are first order in the nucleophile. The nucleophilic rate constants (k_N) obtained are pH independent for all the reactions studied. The Brønsted‐type plot (log k_N vs. pK_a nucleophile) obtained for the phenolysis is linear with slope β=0.21; no break was found at pK_a 7.5, consistent with a concerted mechanism. The Brønsted‐type plots for the SA aminolysis and pyridinolysis are linear with slopes β=0.39 and 0.43, respectively, also suggesting concerted processes. The concerted mechanisms for the latter reactions are proposed on the basis of the lack of break in the Brønsted‐type plots and the instability of the hypothetical pentacoordinate intermediates formed in these reactions. © 2011 Wiley Periodicals, Inc. Int J Chem Kinet 43: 708–714, 2011     

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

A useful ²J(N?H) coupling‐based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA‐binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T‐cell intracellular antigen‐1 (TIA‐1) protein. The pK_a values of the His96 ionizable groups were substantially higher in the complexes with short U‐rich RNA and T‐rich DNA oligonucleotides than those of the isolated TIA‐1 RRM2. Herein, the methodology applied to determine changes in pK_a of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA‐binding proteins that shuttle among different cellular compartments.     

Modeling of serine protease prototype reactions with the flexible effective fragment potential quantum mechanical/molecular mechanical method

A complete cycle of chemical transformations for the serine protease prototype reaction is modeled following calculations with the flexible effective fragment quantum mechanical/molecular mechanical (QM/MM) method. The initial molecular model is based on the crystal structure of the trypsin–bovine pancreatic trypsin inhibitor complex including all atoms of the enzyme within approximately 15–18 Å of the oxygen center O of the catalytic serine residue. Several selections of the QM/MM partitioning are considered. Fractions of the side chains of the residues from the catalytic triad (serine, histidine and aspartic acid) and a central part of a model substrate around the C–N bond to be cleaved are included into the QM subsystem. The remaining part, or the MM subsystem, is represented by flexible chains of small effective fragments, whose potentials explicitly contribute to the Hamiltonian of the QM part, but the corresponding fragment–fragment interactions are described by the MM force fields. The QM/MM boundaries are extended over the C–C bonds of the peptides assigned to the QM subsystem in the enzyme, C–C and C–N bonds in model substrates. Multiple geometry optimizations have been performed by using the RHF/6-31G method in the QM part and OPLSAA or AMBER sets of MM parameters, resulting in a series of stationary points on the complex potential-energy surfaces. All structures generally accepted for the serine protease catalytic cycle have been located. Energies at the stationary points found have been recomputed at the MP2/6-31+G^* level for the QM part in the protein environment. Structural changes along the reaction path are analyzed with special attention to hydrogen-bonding networks. In the case of a model substrate selected as a short peptide CH₃(NHCO-CH₂)₂ – HN–CO–(CH₂–NHCO)CH₃ the computed energy profile for the acylation step shows too high activation energy barriers. The energetics of this rate-limiting step is considerably improved, if more realistic model for the substrate is considered, following the motifs of the ThrI11–GlyI12–ProI13-–CysI14–LysI15–AlaI16–ArgI17–IleI18–IleI19 sequence of the bovine pancreatic trypsin inhibitor.     

Kinetics and Protein‐Inhibitor Docking Studies of Enantiomers of exo‐2‐Norbornyl‐N‐n‐butylcarbamates as Pseudomonas Lipase Inhibitors to Probe the Enantioselectivity of the Enzyme

The Pseudomonas species lipase inhibition shows enantioselectivity for R‐enantiomer over S‐enantiomer of exo‐2‐norbornyl‐N‐n‐butylcarbamates. R‐, S‐, and racemic‐exo‐2‐norbornyl‐N‐n‐butylcarbamates are all characterized as pseudo substrate inhibitors of the enzyme. Thus, the mechanism for Pseudomonas species lipase‐catalyzed hydrolysis of the inhibitor is formation of the first enzyme‐inhibitor Michaelis complex via nucleophilic attack of the active site serine to the inhibitor (K_i step) then formation of the butylcarbamyl enzyme intermediate from this complex (k₂ step). Comparison of bimolecular rate constants (k_i = k₂ / K_i) of the inhibitors indicates that R‐enantiomer is 1.8 times more potent than S‐enantiomer. Thus, Pseudomonas species lipase shows enantioselectivity of 1.8 for R‐exo‐2‐norbornyl‐N‐n‐butyl‐carbamate over S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate. Protein‐ligand interaction studies on both enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate as inhibitors of Pseudomonas species lipase using AutoDock suggest that R‐enantiomer binds more tightly into the active site of the enzyme than S‐enantiomer. The norbornyl ring of S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate is repulsive to Ser 82 and His 251 of the catalytic triad as well as to Met 16 of the oxyanion hole. These repulsions may create few unfavorable interactions between S‐exo‐2‐norbornyl‐N‐n‐butylcarbamate and the enzyme and make this inhibitor a less potent one.     

PKA17—A Coarse-Grain Grid-Based Methodology and Web-Based Software for Predicting Protein pK a Shifts

We have developed and tested PKA17, a coarse-grain grid-based model for predicting protein pK _a shifts. Our pK _a predictor is currently deployed via a website interface. We have carried out parameter fitting using 442 Asp, Glu, His, Lys, and Arg residues for which experimental results are available in the literature. PROPKA software has been used for benchmarking. The average unsigned error and root-mean-square deviation (RMSD) have been found to be 0.628 and 0.831 pH units, respectively, for PKA17. The corresponding results with PROPKA are 0.761 and 1.063 units. We have assessed the robustness of the developed PKA17 methodology with a number of tests and have also explored the possibility of using a combination of PROPKA and PKA17 calculations in order to improve the accuracy of predicted pK _a values for protein residues. We have also once again confirmed that protein acidity constants are influenced almost entirely by residues in the immediate spatial proximity of the ionizable amino acids. The resulting PKA17 software has been deployed online with a web-based interface at http://users.wpi.edu/~jpcvitkovic/pka_calc.html . © 2019 Wiley Periodicals, Inc.     

Potentiometric and Voltammetric Study of 6‐Benzylaminopurine and Its Derivatives

The protonation‐deprotonation equilibrium of 6‐benzylaminopurine (6‐BAP) and its derivatives was studied by potentiometry and voltammetry. The effect of Cl‐ or OCH₃‐group in position 2′, 3′ and 4′ of the benzene ring of 6‐BAP on both pK_a values was investigated. To determine the enthalpy and entropy, the temperature dependence of pK_a was employed. It was found that with increasing temperature the pK_a decreased. In comparison with 6‐BAP the chloro‐ or methoxy‐ group resulted in pK_a increase. The first pK_a values were also determined by linear sweep (LSV) and elimination voltammetry with linear scan (EVLS). New approaches were shown not only for the determination of pK_a from voltammetric titration curves but also for the evaluation of the reduction processes of benzylaminopurines.     

MCCE2: Improving protein pKa calculations with extensive side chain rotamer sampling

Multiconformation continuum electrostatics (MCCE) explores different conformational degrees of freedom in Monte Carlo calculations of protein residue and ligand pK_as. Explicit changes in side chain conformations throughout a titration create a position dependent, heterogeneous dielectric response giving a more accurate picture of coupled ionization and position changes. The MCCE2 methods for choosing a group of input heavy atom and proton positions are described. The pK_as calculated with different isosteric conformers, heavy atom rotamers and proton positions, with different degrees of optimization are tested against a curated group of 305 experimental pK_as in 33 proteins. QUICK calculations, with rotation around Asn and Gln termini, sampling His tautomers and torsion minimum hydroxyls yield an RMSD of 1.34 with 84% of the errors being <1.5 pH units. FULL calculations adding heavy atom rotamers and side chain optimization yield an RMSD of 0.90 with 90% of the errors <1.5 pH unit. Good results are also found for pK_as in the membrane protein bacteriorhodopsin. The inclusion of extra side chain positions distorts the dielectric boundary and also biases the calculated pK_as by creating more neutral than ionized conformers. Methods for correcting these errors are introduced. Calculations are compared with multiple X‐ray and NMR derived structures in 36 soluble proteins. Calculations with X‐ray structures give significantly better pK_as. Results with the default protein dielectric constant of 4 are as good as those using a value of 8. The MCCE2 program can be downloaded from http://www.sci.ccny.cuny.edu/～mcce . © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Catalytic hydrolysis of p‐nitrophenyl picolinate by copper(II) and zinc(II) complexes of N‐(2‐deoxy‐β‐D‐glucopyranosyl‐2‐salicylaldimino)

D‐glucosamine Schiff base N‐(2‐deoxy‐β‐D‐glucopyranosyl‐2‐salicylaldimino) and its Cu(II) and Zn(II) complexes were synthesized and characterized. The hydrolysis of p‐nitrophenyl picolinate (PNPP) catalyzed by ligand and complexes was investigated kinetically by observing the rates of the release of p‐nitrophenol in the aqueous buffers at 25°C and different pHs. The scheme for reaction acting mode involving a ternary complex composed of ligand, metal ion, and substrate was established and the reaction mechanisms were discussed by metal–hydroxyl and Lewis acid mechanisms. The experimental results indicated that the complexes, especially the Cu(II) complex, efficiently catalyzed the hydrolysis of PNPP. The catalytic reactivity of the Zn(II) complex was much smaller than the Cu(II) complex. The rate constant k_N showing the catalytic reactivity of the Cu(II) complex was determined to be 0.299 s^?1 (at pH 8.02) in the buffer. The pK_a of hydroxyl group of the ternary complex was determined to be 7.86 for the Cu(II) complex. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 345–350, 2002     

Tunable Probes with Direct Fluorescence Signals for the Constitutive and Immunoproteasome

Electrophiles are commonly used for the inhibition of proteases. Notably, inhibitors of the proteasome, a central determinant of cellular survival and a target of several FDA‐approved drugs, are mainly characterized by the reactivity of their electrophilic head groups. We aimed to tune the inhibitory strength of peptidic sulfonate esters by varying the leaving groups. Indeed, proteasome inhibition correlated well with the pK_a of the leaving group. The use of fluorophores as leaving groups enabled us to design probes that release a stoichiometric fluorescence signal upon reaction, thereby directly linking proteasome inactivation to the readout. This principle could be applicable to other sulfonyl fluoride based inhibitors and allows the design of sensitive probes for enzymatic studies.     

Capillary electrokinetic fractionation mass spectrometry (CEkF/MS): Technology setup and application to metabolite fractionation from complex samples coupled at‐line with ultrahigh‐resolution mass spectrometry

Capillary electrokinetic fractionation (CEkF) is investigated as a new, simple, and robust approach for semipreparative and analytical sample analysis based on pK_a‐dependant pH‐driven electrophoretic mobility. CEkF was optimized with contactless conductivity detection and conducted with 10 kV reverse voltage for 10 min, then coupled on/at‐line to ESI/MS. We propose a semi‐empirical model with 14 representative compounds based on the correlation between sample/medium pH regulating the partial charge, the electrokinetic loading of the capillary and intensity (I) of analytes. According to the model, an empirical function (I = f (pH)) could be derived to calculate the acid dissociation constant (pK_a) of various model compounds based on their pH‐dependant MS intensity profiles with the RSD < 4.05. Using the ultrahigh‐resolution of ion cyclotron resonance Fourier transform MS, the pK_a model was further illustrated in real samples into the structure prediction of important compounds in wine over two vintages. The established CEkF was successfully used to selectively fractionate sulfur compounds from the complex wine samples at pH 1.66. The proposed CEkF approach should allow in the future the simultaneous pK_a evaluation of multiple constituents without complicated separation out of a complex mixture in metabolomics or environmental chemistry.     

Accurate pKa Calculation of the Conjugate Acids of Alkanolamines,Alkaloids and Nucleotide Bases by Quantum Chemical Methods

The pK_a of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08‐HX and M11‐L) and ab initio methods (SCS‐MP2, G3). Implicit solvent effects are included with a conductor‐like polarizable continuum model (CPCM) and universal solvation models (SMD, SM8). G3, SCS‐MP2 and M11‐L methods coupled with SMD and SM8 solvation models perform well for alkanolamines with mean unsigned errors below 0.20 pK_a units, in all cases. Extending this method to the pK_a calculation of 35 nitrogen‐containing compounds spanning 12 pK_a units showed an excellent correlation between experimental and computational pK_a values of these 35 amines with the computationally low‐cost SM8/M11‐L density functional approach.