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.     

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.     

NADH interactions with WT- and S94A-acyl carrier protein reductase from Mycobacterium tuberculosis: an ab initio study

We present an ab initio molecular dynamics study of the complex between acyl carrier protein reductase InhA from M. tuberculosis and isonicotinic acid hydrazide-NADH. We focus on wild-type (WT) InhA and a mutant causing drug resistance (S94A) for which structural information is available (Rozwarski et al., 1998;279:98--102; Dessen et al., 1995;267:1638--1641). Our calculations suggest that the water-mediated H-bond interactions between Ser94 side chain and NADH, present in WT InhA X-ray structure, can be lost during the dynamics. This conformational change is accompanied by a structural rearrangement of Gly14. The calculated structure of WT is rather similar to the X-ray structure of the S94A mutant in terms of geometrical parameters and chemical bonding. Further evidence for the mobility of Ser94 is provided by a 1-ns-long classical molecular dynamics on the entire protein. The previously unrecognized high mobility of Ser94 can provide a rationale of the small change in free binding energies on passing from WT to S94A InhA.     

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.     

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 nature of intermolecular interactions between aromatic amino acid residues

The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single-point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic-aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures. Proteins 2002;48:117-125.     

The deuterium isotope effect on the NMR signal of the low-barrier hydrogen bond in a transition-state analog complex of chymotrypsin

The participation of a low-barrier hydrogen bond (LBHB) in the mechanism of action of chymotrypsin introduces a new role for Asp 102 and His 57 in catalysis [C. S. Cassidy, J. Lin, and P. A. Frey (1997) Biochemistry 36, 4576-4584]. It is postulated that the LBHB increases the basicity of His 57-N(epsilon2) in the transition state, thereby facilitating the abstraction of a proton from Ser 195, and stabilizes the tetrahedral intermediate in the acylation step. Evidence for this mechanism includes the downfield chemical shift of the proton bridging His 57 and Asp 102 in transition-state analog complexes and the low deuterium fractionation factors for this proton in the same complexes. We present additional spectroscopic evidence supporting the assignment of an LBHB between His 57 and Asp 102. The tetrahedral addition complex between Ser 195 of chymotrypsin and N-acetyl-l-leucyl-l-phenylalanyl trifluoromethylketone is regarded as a close structural analog of a tetrahedral intermediate. The deuterium NMR signal for the downfield deuteron bridging His 57 and Asp 102 in D(2)O has now been observed as a broad band centered at 17.8 +/- 0.5 ppm. The proton NMR signal in H(2)O is centered at 18.9 +/- 0.05 ppm. The two signals are clearly separated corresponding to a deuterium isotope effect of Delta[delta(H) - delta(D)] = 1.1 +/- 0.5 ppm. Deuterium isotope effects in this range are characteristic of LBHBs, and this observation provides further support for the assignment of the proton bridging His 57 and Asp 102 in transition-state analog complexes as an LBHB.     

Conformational flexibility of the catalytic Asp dyad in HIV-1 protease: An ab initio study on the free enzyme

The enzyme protease from the human immunodeficiency virus type 1 (HIV-1 PR) is one of the main targets for therapeutic intervention in AIDS. Computer modeling is useful for probing the binding of novel ligands, yet empirical force field-based methods have encountered problems in adequately describing interactions of the catalytic aspartyl pair. In this work we use ab initio dynamic methods to study the molecular interactions and the conformational flexibility of the Asp dyad in the free enzyme. Calculations are performed on model complexes that include, besides the Asp dyad, the conserved Thr26 and Gly27 residues and water molecules present in the active site channel. Our calculations provide proton location and binding mode of the active-site water molecule, which turn out to be different from those of the eukariotic isoenzyme. Furthermore, the calculations reproduce well the structural features of the aspartyl dyad in the protein. Finally, they allow the identification of both dipole/charge interactions and a low-barrier hydrogen bond as important stabilizing factors for the peculiar conformation of the active site. These findings are consistent with site-directed mutagenesis experiments on the 27, 27; positions (Bagossi et al., Protein Eng 1996;9:997-1003). The electric field of the protein frame (included in some of the calculations) does not affect significantly the chemical bonding at the cleavage site. Proteins 2000;39:26-36.     

Contribution of cation-pi interactions to the stability of protein-DNA complexes

Cation-pi interactions between an aromatic ring and a positive charge located above it have proven to be important in protein structures and biomolecule associations. Here, the role of these interactions at the interface of protein-DNA complexes is investigated, by means of ab initio quantum mechanics energy calculations and X-ray structure analyses. Ab initio energy calculations indicate that Na ions and DNA bases can form stable cation-pi complexes, whose binding strength strongly depends on the type of base, on the position of the Na ion, and whether the base is isolated or included in a double-stranded B-DNA. A survey of protein-DNA complex structures using appropriate geometrical criteria revealed cation-pi interactions in 71% of the complexes. More than half of the cation-pi pairs involve arginine residues, about one-third asparagine or glutamine residues that only carry a partial charge, and one-seventh lysine residues. The most frequently observed pair, which is also the most stable as monitored by ab initio energy calculations, is arginine- guanine. Arginine-adenine interactions are also favorable in general, although to a lesser extent, whereas those with thymine and cytosine are not. Our calculations show that the major contribution to cation-pi interactions with DNA bases is of electrostatic nature. These interactions often occur concomitantly with hydrogen bonds with adjacent bases; their strength is estimated to be from three to four times lower than that of hydrogen bonds. Finally, the role of cation-pi interactions in the stability and specificity of protein-DNA complexes is discussed.     

The role of the environment around the catalytic triad in β-trypsin: A molecular orbital study

In the enzymatic reaction of β-trypsin the role of environment around the catalytic triad is studied by means of ab initio molecular orbital calculations. The triple ion form of the catalytic triad (Asp 102(?)-His 57(+)-Ser 195(?)) is considerably more stable than the double proton-transferred form (Asp 102(neutral)-His 57(neutral)-Ser 195(?)), due to the environment around it, rather than its nature. The “electrostatic mechanism” is more favorable than the “charge relay mechanism” owing to the nature of the enzyme as a biopolymer.     

Structure and electronic properties of heme complexes with various ligands

The electronic and atomic structures, and the molecular dynamics of the atomic structure at 310 K of a set of heme complexes with His and Gly amino acids in the 5th coordination position and some ligands (O2, NO) in the 6th position were studied by ab initio (3-21G basis set) and semiempirical (PM3) quantum chemistry methods and the method of molecular dynamics. It was shown that the type of coordination of the imidazole ring influences the constant of chemical bonding of molecular oxygen of the complexes. On the other hand, NO and O2 molecules have different transinfluence on the ligand in the 5th coordination position. It was shown that temperature affects profoundly the atomic and electronic structures of the complexes, the tightness of chemical bonding and their reactivity.     

Role of Asp102 in the enzymatic reaction of bovine β-trypsin: A molecular orbital study

Using the X-ray structure of the complex of bovine β-trypsin with the basic pancreatic trypsin inhibitor, the hydrogen-bond structure consisting of Ser195, His57 and Asp102 is clarified in relation to the mechanism of the enzymatic reaction from an ab initio quantum chemical point of view. Under the influence of the inhibitor, of the three hydrogen bonds involving Ser214, His57 and Ala56 around Asp102, and of the other ionic amino acid residues, Asp102 plays a significant role in lowering the barrier height of the proton transfer from Ser195 to His57 without accepting a proton from His57. The principal cause of the barrier height lowering is the electrostatic interaction.     

Coherent singlet-triplet transitions at the initial step of tubulin assembly into microtubules

Quantum chemistry calculations [DFT-B3LYP QM/MM method, 6-31G** basis set, + ab initio molecular dynamics] were used to study the action of Mg2+ on tubulin properties. It was shown that the hydration of the guanosine triphosphate-tubulin forms a protein zone structure, which includes a electron-occupied zone and a conductivity zone. The binding of Mg2+ to guanosine triphosphate-tubulin results in the unpairing of electrons in the occupied zone (triplet state formation) followed by their transition to the conductivity zone in which the inversion of spin occurs (singlet state formation). The formation of triplet state is the initial step in the subsequent protein dynamics in the picosecond range of time. The dynamics shows up as a coherent oscillating transition of tubulin between the triplet and singlet states, which is evidence of a simultaneous adjustment between nuclear and electron configurations of the protein (ab initio molecular dynamics calculations). The barrier between the triplet and singlet states does not exceed 0.60 kcal x mol(-1). The barrier overcome is considered as electron tunneling through the Fermi surface, which separates the occupied and conductivity zones. Zone formation occurs in the presence of the shell of biological water surrounding the protein.     

How does activation loop phosphorylation modulate catalytic activity in the cAMP-dependent protein kinase: a theoretical study

Phosphorylation mediates the function of many proteins and enzymes. In the catalytic subunit of cAMP-dependent protein kinase, phosphorylation of Thr 197 in the activation loop strongly influences its catalytic activity. In order to provide theoretical understanding about this important regulatory process, classical molecular dynamics simulations and ab initio QM/MM calculations have been carried out on the wild-type PKA-Mg(2) ATP-substrate complex and its dephosphorylated mutant, T197A. It was found that pThr 197 not only facilitates the phosphoryl transfer reaction by stabilizing the transition state through electrostatic interactions but also strongly affects its essential protein dynamics as well as the active site conformation.     

Molecular dynamics study on drug resistance mechanism of HCV NS3/4A protease inhibitor: BI201335

Hepatitis C virus (HCV) infection is a serious threat to global health. NS3 serine protease is one of the most advanced HCV drug targets. However, the high mutation rate makes many protease inhibitors ineffective and allows viral replication to continue. To investigate the structural basis of the molecular mechanism of HCV resistance to inhibitors, molecular dynamics and molecular mechanics Poisson–Boltzmann/surface area calculations were carried out on HCV NS3 serine protease–BI201335 complexes. The drug resistance to BI201335 is explained by the fact that seven single mutations weaken the biological activity by lessening the sum of electrostatic interactions in the gas phase and polar solvation. The computational results demonstrate that the mutations affect the BI201335 binding through direct and indirect mechanisms. Seven single mutations lead to significant changes in the conformation, such as the shifts of the side chain of His57 and Lys136 and the movement of the P2 group of BI201335 towards the solvent. Furthermore, the contributions of Lys136 significantly decrease, which is the most major binding attraction. The shifts of the side chain of His57 induce the lack of hydrogen bond between His57 with Asp81 expert for D168G mutation. Detailing the molecular mechanisms of BI201335 drug resistance provides some helpful insights into the nature of mutational effect and aid the rational design of potent inhibitors combating HCV.     

Computer-aided molecular design of 1H-imidazole-2,4-diamine derivatives as potential inhibitors of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> DHFR enzyme

Design and discovery of new potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), equally active against both the wild-type and mutant strains, is urgently needed. In this study, a computer-aided molecular design approach that involved ab initio molecular orbital and density functional theory calculations, along with molecular electrostatic potential analysis, and molecular docking studies was employed to design 15 1H-imidazole-2,4-diamine derivatives as potential inhibitors of PfDHFR enzyme. Visual inspection of the binding modes of the compounds demonstrated that they all interact, via H-bond interactions, with key amino acid residues (Asp54, Ileu/Leu164, Asn/Ser108 and Ile14) similar to those of WR99210 (3) in the active site of the enzymes used in the study. These interactions are known to be essential for enzyme inhibition. These compounds showed better or comparable binding affinities to that of the bound ligand (WR99210). In silico toxicity predictions, carried out using TOPKAT software, also indicated that the compounds are non-toxic.     

Crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 A resolution: dynamics of catalytic residues

We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C(alpha)-C(beta)-C(gamma) angle (126 degrees ) that deviated by 13.4 degrees from the "ideal" bond angle of 112.6 degrees. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C(alpha)-C(beta)-C(gamma) angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N(delta)(1) and Asp108 in conformation A gave the most exothermic reaction (DeltaH = -22 kcal/mol). With His272 doubly protonated at N(delta)(1) and N(epsilon)(2), the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C(alpha)-C(beta)-C(gamma) angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.     

Factors determining the relative stability of anionic tetrahedral complexes in serine protease catalysis and inhibition

Quantum mechanical ab initio (RHF/6-31+G*//RHF/3-21G) calculations were used to simulate the formation of the tetrahedral complex intermediate (TC) in serine protease active site by substrates and transition-state analog inhibitors. The enzyme active site was simulated by an assembly of the amino acids participating in catalysis, whereas the substrates and inhibitors were simulated by small ligands, acetamide (1) and trifluoroacetone (2), respectively. For the first time, the principal factors determining the relative stability of the TC in serine proteases are arranged according to their energy contributions. These include (a) formation of the new covalent bond between Ser195 O(gamma) and the electrophilic center of a ligand; (b) stabilization of the oxyanion in the oxyanion hole; (c) basic catalysis by His57; and (d) hydrogen bond between Asp102 carboxylate and N(delta) of the protonated His57. We have directly calculated the gas-phase relative free energy of formation of TC(AS)(2) and TC(AS)(1), the value of DeltaDeltaG(g)[TC(AS)(2,1)]. It is DeltaE(cov), the relative energy of the new covalent bond between the enzyme and the ligand formed in a TC that determines the experimentally observed large difference in the stability of TCs formed by substrates and TS-analog inhibitors of serine proteases. We demonstrated that the relative stability of TCs formed by a series of mono- and dipeptide amides and TFKs, derived from experimental kinetic data, can be rather well approximated by the sum of the theoretically calculated value of DeltaDeltaG(g)[TC(AS)(2, 1)] and the difference in hydration free energies of isolated ligands.