Low-barrier hydrogen bond hypothesis in the catalytic triad residue of serine proteases: correlation between structural rearrangement and chemical shifts in the acylation process

To elucidate the catalytic advantage of the low-barrier hydrogen bond (LBHB), we analyze the hydrogen bonding network of the catalytic triad (His57-Asp102-Ser195) of serine protease trypsin, one of the best examples of the LBHB reaction mechanism. Especially, we focus on the correlation between the change of the chemical shifts and the structural rearrangement of the active site in the acylation process. To clarify LBHB, we evaluate the two complementary properties. First, we calculate the NMR chemical shifts of the imidazole ring of His57 by the gauge-including atomic orbital (GIAO) approach within the ab initio QM/MM framework. Second, the free energy profile of the proton transfer from His57 to Asp102 in the tetrahedral intermediate is obtained by ab initio QM/MM calculations combined with molecular dynamics free energy perturbation (MD-FEP) simulations. The present analyses reveal that the calculated shifts reasonably reproduce the observed values for (1)H chemical shift of H(epsilon)(1) and H(delta)(1) in His57. The (15)N and (13)C chemical shifts are also consistent with the experiments. It is also shown that the proton between His57 and Asp102 is localized at the His57 side. This largely downfield chemical shift is originated from the strong electrostatic interaction, not a covalent-like bonding character between His57 and Asp102. Also, it is proved that a slight downfield character of H(epsilon)(1) is originated from a electrostatic interaction between His57 and the backbone carbonyl group of Val213 and Ser214. These downfield chemical shifts are observed only when the tetrahedral intermediate is formed in the acylation process.     

Role of catalytic residues in the formation of a tetrahedral adduct in the acylation reaction of bovine β-trypsin: A molecular orbital study

In the acylation reaction of serine proteases the effect of amino acid residues on the geometrical change of the catalytic site from Michaelis to tetrahedral state was studied by using ab initio molecular orbital calculations. Amino acid residues in the catalytic site and the peptide substrate were calculated as a quantum mechanical region, and all the other amino acid residues and the calcium ion were included in the calculation as the electrostatic effects. The effects of Asp102, Asp194, N-terminus and the oxyanion binding site are large. The oxyanion binding site directly stabilizes the tetrahedral substrate. Asp102 stabilizes the enzyme intermediate, interacting with the protonated His57 residue. In order to elucidate the roles of Asp102 and the oxyanion binding site, energy decomposition analyses were done for the intermolecular interactions. The contribution of Asp102 and the oxyanion binding site to the decrease of energy in the geometrical change is due to the electrostatic effect. The energies of the proton shuttle from Ser195 O^γ to the leaving group of the substrate were calculated for amide and ester substrate models.     

The pKa value of His 57-Asp 102 couple in the active site of bovine pancreatic beta-trypsin: a molecular orbital study

The charge relay hypothesis generated a large number of theoretical and experimental studies that tested the ideas involved. Opinion based upon theoretical and experimental studies is divided on the prediction, although there are many experimental data which do not support the hypothesis. The essential feature is the proton transfer from the histidine imidazole to the aspartate. Thus, we have performed the detailed calculations of the proton transfer from His 57 to Asp 102 including the environment of the couple in protonated bovine pancreatic β-trypsin. The charge state of the His 57-Asp 102 couple is greatly influenced by the environment of the enzyme around it. In this paper, it is shown that the proton between His 57 and Asp 102 is covalently bonded to the His 57 imidazole in the protonated β-trypsin. Our MO calculations, which support the neutral-pK-histidine theory as the results, do not support the charge relay mechanism.     

QM/MM study of the active site of free papain and of the NMA-papain complex.

Hybrid quantum mechanical/molecular mechanical (QM/MM) calculations using restricted and unrestricted Hartree-Fock and B3LYP ab initio (QM) and Amber force field (MM), respectively, have been applied to study the catalytic site of papain in both free and substrate bonded forms. Ab initio geometry optimizations have been performed for the active site of papain and the N-methyl-acetamide (NMA)-papain complex within the molecular mechanical treatment of the protein environment. A covalent tetrahedral intermediate structure could be obtained only when the amide N atom of the substrate molecule was protonated through a proton transfer from the His-159 in the catalytic site. Our results support the previous assumption that a proton transfer from His-159 to the amide N atom of the substrate occurs prior to or concerted with the nucleophilic attack of the Cys-25 sulfur atom to the carbonyl group of the substrate. The electron correlation effect will reduce the proton transfer barrier. Therefore, this proton transfer can be easily observed in the B3LYP/6-31G* calculations. The HF/6-31G* method overestimates the reaction barrier against this proton transfer. The sulfur atom of Cys-25 and the imidazole ring of His-159 are found to be coplanar in the free form of the enzyme. However, the rotation of the imidazole ring of His-159 was observed during the formation of the tetrahedral intermediate. Without the papain environment, the coplanar thiolate-imidazolium ion pair RS-...ImH+ is much less stable than the neutral form of RSH....Im. Within the protein environment, however, the thiolate-imidazolium ion pair becomes more stable than its neutral form by 4.1 and 0.4 kcal/mol in HF/6-31G* and B3LYP/6-31G* calculations, respectively. The barrier of proton transfer from S-H group of Cys-25 to the imidazole ring of His-159 was reduced from 22.0 kcal/mol to 15.2 kcal/mol by the protein environment in HF/6-31G* calculations. This barrier is found to be much smaller (2.5 kcal/mol) in B3LYP/6-31G* calculations.     

Density functional theory study on the interaction between keto-9H guanine and aspartic acid

A theoretical study was performed using density functional theory (DFT) to investigate hydrogen bonding interactions in signature complexes formed between keto-9H guanine (Gua) and aspartic acid (Asp) at neutral pH. Optimized geometries, binding energies and the theoretical IR spectra of guanine, aspartic acid and their corresponding complexes (Gua-Asp) were calculated using the B3LYP method and the 6-31+G(d) basis set. Stationary points found to be at local minima on the potential energy surface were verified by second derivative harmonic vibrational frequency calculations at the same level of theory. AIM theory was used to analyze the hydrogen bonding characteristics of these DNA base complex systems. Our results show that the binding motif for the most stable complex is strikingly similar to a Watson-Crick motif observed in the guanine-cytosine base pair. We have found a range of hydrogen bonding interactions between guanine and aspartic acid in the six complexes. This was further verified by theoretical IR spectra of ω(C-H---O-H) cm⁻¹ stretches for the Gua-Asp complexes. The electron density plot indicates strong hydrogen bonding as shown by the 2p _z dominant HOMO orbital character.     

Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or “site.” Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment–proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment–protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna–Mathews–Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.     

Hydrogen bonding in the active site of ketosteroid isomerase: electronic inductive effects and hydrogen bond coupling

Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pK(a) of the phenolate increases, these hydrogen bond distances decrease, the associated nuclear magnetic resonance (NMR) chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond becomes longer and the Asp103-phenolate hydrogen bond shorter, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen bonding network on the NMR chemical shifts and electronic absorption spectra.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site

Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.     

Water Pathways in the Bacteriorhodopsin Proton Pump

Internal water molecules play key roles in the functioning of the light-driven bacteriorhodopsin proton pump. Of particular importance is whether during the proton-pumping cycle the critical water molecule w402 can relocate from the extracellular to the cytoplasmic side of the retinal Schiff base. Here, classical mechanical and combined quantum mechanical/molecular mechanical reaction path computations are performed to investigate pathways and energetic factors influencing w402 relocation. Hydrogen bonding between w402 and the negatively charged Asp85 and Asp212 largely opposes repositioning of the water molecule. In contrast, favorable contributions from hydrogen bonding of w402 with the Schiff base and Thr89 and from the untwisting of the retinal polyene chain lower the energetic cost for water relocation. The delicate balance between the competing contributions underlies the need for highly accurate calculations and structural information.     

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane.

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.     

Is there a weak H‐bond → LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.     

Prodomain processing of Asp1 (BACE2) is autocatalytic

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. The highly elusive beta-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit beta-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu(62) and Ala(63) and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P(1)' and P(2)' residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells.     

Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings

We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N(epsilon2) coordination bond, and protonation of the N(delta1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N(epsilon2) position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N(epsilon1) position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N(delta1) atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N(epsilon2) atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.     

Modification of histidine (B10) is the causative agent for a superactive form of insulin.

The site of modification that is responsible for the formation of superactive insulin (ILM) was determined. The insulin derivative was prepared by treatment of insulin-Sepharose with ammonium bicarbonate. It was found that the insulin was bound to the resin through histidine B10, His (B10), and its ammonium bicarbonate-mediated release resulted in an insulin analog in which His (B10) was modified on the imidazole ring. This modification was reversible upon storage, resulting in normal levels of insulin activity. Amino acid analysis of a peptide containing this modified histidine revealed some aspartic acid. Since Asp (B10) insulin is also superactive, the observed superactivity may thus stem from either modification of the histidine or its conversion to aspartic acid.     

Insights into the structure and dynamics of the dinuclear zinc beta-lactamase site from Bacteroides fragilis

Herein, we report quantum chemical calculations and molecular dynamics (MD) simulations of the dinuclear form of the Bacteroides fragilis zinc beta-lactamase. We studied four different configurations which differ in the protonation state of the Asp103 residue and in the presence or absence of a Zn1-OH-Zn2 bridge. The flexibility of the Zn1-OH-Zn2 bridge was studied by means of quantum mechanical (QM) calculations on cluster models while the relative stabilities of the different configurations were estimated from QM linear scaling calculations on the enzyme. Contacts between important residues (Cys104, Asp69, Lys185, etc.), the solvation of the zinc ions, and the conformation of the active site beta-hairpin loop were characterized by the MD analyses. The influence of the buried sodium ion close to the Zn2 position was investigated by carrying out a secondary simulation where the sodium ion was replaced with an internal water molecule. The comparative structural analyses among the different MD trajectories augmented with energetic calculations have demonstrated that the B. fragilis protein efficiently binds the internal Na(+) ion observed crystallographically. Moreover, we found that when Asp103 is unprotonated, a rigid Zn1-OH-Zn2 bridge results, while for neutral Asp103, a fluctuating Zn1-Zn2 distance was possible via the breaking and formation of the Zn1-OH-Zn2 bridge. The mechanistic implications of these observations are discussed in detail.     

Analysis of the factors that influence the C=N stretching frequency of polyene Schiff bases. Implications for bacteriorhodopsin and rhodopsin.

In this study quantum mechanical calculations of force constants and normal mode analysis are used to elucidate the factors that influence the C=C and C=N stretching frequencies in polyenes and in protonated Schiff bases. The C=N stretching frequency is found to depend on both the C=N stretching force constant and the C=N-H bending force constant. Due to the contributions of these two modes, the C=N stretching frequency is particularly sensitive to the magnitude of the Schiff base counterion interactions and to the hydrogen bonding environment of the Schiff base nitrogen. Models for chromophore-protein interactions in the retinal binding site and for the photochemical transformations of bacteriorhodopsin and rhodopsin are evaluated in light of these results.     

X-ray crystal structure of the ferric sperm whale myoglobin: imidazole complex at 2.0 A resolution

The X-ray crystal structure of the ferric sperm whale (Physeter catodon) myoglobin:imidazole complex has been refined at 2.0 A resolution, to a final R-factor of 14.8%. The overall conformation of the protein is little affected by binding of the ligand. Imidazole is co-ordinated to the heme iron at the distal site, and forces distinguishable local changes in the surrounding protein residues. His64(E7) swings out of the distal pocket and becomes substantially exposed to the solvent: nevertheless, it stabilizes the exogenous ligand by hydrogen bonding. The side-chains of residues Arg45(CD3) and Asp60(E3) are also affected by imidazole association.