Theoretical study of the mechanisms of substrate recognition by catalase

A variety of theoretical methods including classical molecular interaction potentials, classical molecular dynamics, and activated molecular dynamics have been used to analyze the substrate recognition mechanisms of peroxisomal catalase from Saccharomyces cerevisiae. Special attention is paid to the existence of channels connecting the heme group with the exterior of the protein. On the basis of these calculations a rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations. According to our calculations the water is expected to be a competitive inhibitor of the enzyme, blocking the access of hydrogen peroxide to the active site. The main channel is the preferred route for substrate access to the enzyme and shows a cooperative binding to hydrogen peroxide. However, the overall affinity of the main channel for H(2)O(2) is only slightly larger than that for H(2)O. Alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected. These secondary channels might be important for product release.     

What Is the Catalytic Mechanism of Enzymatic Histone N-Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

Arginine methylation is an important mechanism of epigenetic regulation. Some Fe(II) and 2-oxoglutarate dependent Jumonji-C (JmjC) Nϵ-methyl lysine histone demethylases also have N-methyl arginine demethylase activity. We report combined molecular dynamic (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) studies on the mechanism of N-methyl arginine demethylation by human KDM4E and compare the results with those reported for N-methyl lysine demethylation by KDM4A. At the KDM4E active site, Glu191, Asn291, and Ser197 form a conserved scaffold that restricts substrate dynamics; substrate binding is also mediated by an out of active site hydrogen-bond between the substrate Ser1 and Tyr178. The calculations imply that in either C−H or N−H potential bond cleaving pathways for hydrogen atom transfer (HAT) during N-methyl arginine demethylation, electron transfer occurs via a σ-channel; the transition state for the N−H pathway is ∼10 kcal/mol higher than for the C−H pathway due to the higher bond dissociation energy of the N−H bond. The results of applying external electric fields (EEFs) reveal EEFs with positive field strengths parallel to the Fe=O bond have a significant barrier-lowering effect on the C−H pathway, by contrast, such EEFs inhibit the N−H activation rate. The overall results imply that KDM4 catalyzed N-methyl arginine demethylation and N-methyl lysine demethylation occur via similar C−H abstraction and rebound mechanisms leading to methyl group hydroxylation, though there are differences in the interactions leading to productive binding of intermediates.     

Second half-reaction of nitric oxide synthase: computational insights into the initial step and key proposed intermediate

Density functional theory methods have been employed to investigate possible first steps in the second half-reaction of the mechanism of nitric oxide synthases (NOSs). In particular, reactions and complexes formed via transfer of either or both hydrogens of the substrates (NHA) -NHOH group to the Fe-bound O2 were considered. For each of these pathways, the effect of adding an extra electron from tetrahydrobiotperin (H4B) was also examined. The preferred initial pathway involves the simultaneous transfer of both hydrogens of the -NHOH group to the Fe(heme)-O2, without an additional electron, to give the Fe(heme)-HOOH species which lies only marginally higher in energy, 2.5 kcal mol(-1) or less, than the initial bound active site. An alternative mechanism in which only the -NH- proton of the -NHOH group is transferred to the Fe(heme)-O2 to give an Fe(heme)-OOH derivative is found to require only slightly more energy, approximately 2 kcal mol(-1). However, transfer of the proton back to the -NOH nitrogen occurs without a barrier at 298.15 K. Tetrahedral intermediates in which the Fe(heme)-O2 has attached at the guanidinium carbon (C(guan)) of NHA, that is, forms an Fe(heme)-O2-C(guan) link, have also been investigated. All examples of such species considered, that is, with or without hydrogen or electron transfers, lie significantly higher in energy by at least 29.0 kcal mol(-1) than the initial bound active site. Thus, it is suggested that such complexes are not mechanistically feasible. The implications of the present findings for the second half-reaction are also discussed.     

Computational study of ketosteroid isomerase: insights from molecular dynamics simulation of enzyme bound substrate and intermediate

Delta(5)-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroids to their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction follows a two-step general acid-base mechanism starting with Asp38-CO(2)(-) mediated proton abstraction from a sp(3)-hybridized carbon atom, alpha to carbonyl group, providing a dienolate intermediate. In the second step, Asp38-CO(2)H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details of the mechanism have been highly controversial despite several experimental and computational studies of this enzyme. The general acid-base catalysis has been proposed to involve either a catalytic diad or a cooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecond molecular dynamics (MD) simulation of enzyme bound natural substrate (E.S) and enzyme bound intermediate (E.In) solvated in a TIP3P water box. The final coordinates from our MD simulation strongly support the cooperative hydrogen bond mechanism. The MD simulation of E.S and E.In shows that both Tyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogen bonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on E.S --> E.In. We also observe dynamic motions of water moving in and out of the active site in the E.S structures. This free movement of water disappears in the E.In structures. The active site is shielded by hydrophobic residues, which come together and squeeze out the waters from the active site in the E.In complex.     

A DFT study of the mechanism of Ni superoxide dismutase (NiSOD): role of the active site cysteine-6 residue in the oxidative half-reaction

In the present DFT study, the catalytic mechanism of H2O2 formation in the oxidative half-reaction of NiSOD, E-Ni(II) + O2- + 2H+ --> E-Ni(III) + H2O2, has been investigated. The main objective of this study is to investigate the source of two protons required in this half-reaction. The proposed mechanism consists of two steps: superoxide coordination and H2O2 formation. The effect of protonation of Cys6 and the proton donating roles of side chains (S) and backbones (B) of His1, Asp3, Cys6, and Tyr9 residues in these two steps have been studied in detail. For protonated Cys6, superoxide binding generates a Ni(III)-O2H species in a process that is exothermic by 17.4 kcal/mol (in protein environment using the continuum model). From the Ni(III)-O2H species, H2O2 formation occurs through a proton donation by His1 via Tyr9, which relative to the resting position of the enzyme is exothermic by 4.9 kcal/mol. In this pathway, a proton donating role of His1 residue is proposed. However, for unprotonated Cys6, a Ni(II)-O2- species is generated in a process that is exothermic by 11.3 kcal/mol. From the Ni(II)-O2- species, the only feasible pathway for H2O2 formation is through donation of protons by the Tyr9(S)-Asp3(S) pair. The results discussed in this study elucidate the role of the active site residues in the catalytic cycle and provide intricate details of the complex functioning of this enzyme.     

Oxygen Migration Pathways in NO‐bound Truncated Hemoglobin

Atomistic simulations of dioxygen (O₂) dynamics and migration in nitric oxide‐bound truncated Hemoglobin N (trHbN) of Mycobacterium tuberculosis are reported. From more than 100 ns of simulations the connectivity network involving the metastable states for localization of the O₂ ligand is built and analyzed. It is found that channel I is the primary entrance point for O₂ whereas channel II is predominantly an exit path although access to the protein active site is also possible. For O₂ a new site compared to nitric oxide, from which reaction with the heme group can occur, was found. As this site is close to the heme iron, it could play an important role in the dioxygenation mechanism as O₂ can remain there for hundreds of picoseconds after which it can eventually leave the protein, while NO is localized in Xe2. The present study supports recent experimental work which proposed that O₂ docks in alternative pockets than Xe close to the reactive site. Similar to other proteins, a phenylalanine residue (Phe62) plays the role of a gate along the access route in channel I. The most highly connected site is the Xe3 pocket which is a “hub” and free energy barriers between the different metastable states are ≈1.5 kcal mol^?1 which allows facile O₂ migration within the protein.     

Hydrogen‐bonding interactions in the active site of a low molecular weight protein‐tyrosine phosphatase

Molecular dynamics simulation of the Michaelis complex, phospho‐enzyme intermediate, and the wild‐type and C12S mutant have been carried out to examine hydrogen‐bonding interactions in the active site of the bovine low molecular weight protein‐tyrosine phosphatase (BPTP). It was found that the S_γ atom of the nucleophilic residue Cys‐12 is ideally located at a position opposite from the phenylphosphate dianion for an inline nucleophilic substitution reaction. In addition, electrostatic and hydrogen‐bonding interactions from the backbone amide groups of the phosphate‐binding loop strongly stabilize the thiolate anion, making Cys‐12 ionized in the active site. In the phospho‐enzyme intermediate, three water molecules are found to form strong hydrogen bonds with the phosphate group. In addition, another water molecule can be identified to form bridging hydrogen bonds between the phosphate group and Asp‐129, which may act as the nucleophile in the subsequent phosphate hydrolysis reaction, with Asp‐129 serving as a general base. The structural difference at the active site between the wild‐type and C12S mutant has been examined. It was found that the alkoxide anion is significantly shifted toward one side of the phosphate binding loop, away from the optimal position enjoyed by the thiolate anion of the wild‐type enzyme in an S_N2 process. This, coupled with the high pK_a value of an alcoholic residue, makes the C12S mutant catalytically inactive. These molecular dynamics simulations provided details of hydrogen bonding interactions in the active site of BPTP, and a structural basis for further studies using combined quantum mechanical and molecular mechanical potential to model the entire dephosphorylation reaction by BPTP. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1192–1203, 2000     

Enzyme Selectivity Fine‐Tuned through Dynamic Control of a Loop

Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand‐selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations.     

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3′-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2′-hydroxy-, 3′-hydroxy- and 4′-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.     

A gate mechanism indicated in the selectivity filter of the potassium channel KscA

Classical molecular dynamics (MD) and non-equilibrium steered molecular dynamics (SMD) simulations were performed on the molecular structure of the potassium channel KcsA using the GROMOS 87 force fields. Our simulations focused on mechanistic and dynamic properties of the permeation of potassium ions through the selectivity filter of the channel. According to the SMD simulations a concerted movement of ions inside the selectivity filter from the cavity to extracellular side depends on the conformation of the peptide linkage between Val76 and Gly77 residues in one subunit of the channel. In SMD simulations, if the carbonyl oxygen of Val76 is positioned toward the ion bound at the S3 site (gate-opened conformation) the net flux of ions through the filter is observed. When the carbonyl oxygen leaped out from the filter (gate-closed conformation), ions were blocked at the S3 site and no flux occurred. A reorientation of the Thr75-Val76 linkage indicated by the CHARMM-based MD simulations performed Berneche and Roux [(2005) Structure 13:591–600; (2000) Biophys J 78:2900–2917] as a concomitant process of the Val76-Gly77 conformational interconversion was not observed in our GROMOS-based MD simulations.     

Electrostatic Perturbations in the Substrate-Binding Pocket of Taurine/α-Ketoglutarate Dioxygenase Determine its Selectivity

Taurine/α-ketoglutarate dioxygenase is an important enzyme that takes part in the cysteine catabolism process in the human body and selectively hydroxylates taurine at the C¹-position. Recent computational studies showed that in the gas-phase the C²−H bond of taurine is substantially weaker than the C¹−H bond, yet no evidence exists of 2-hydroxytaurine products. To this end, a detailed computational study on the selectivity patterns in TauD was performed. The calculations show that the second-coordination sphere and the protonation states of residues play a major role in guiding the enzyme to the right selectivity. Specifically, a single proton on an active site histidine residue can change the regioselectivity of the reaction through its electrostatic perturbations in the active site and effectively changes the C¹−H and C²−H bond strengths of taurine. This is further emphasized by many polar and hydrogen bonding interactions of the protein cage in TauD with the substrate and the oxidant that weaken the pro-R C¹−H bond and triggers a chemoselective reaction process. The large cluster models reproduce the experimental free energy of activation excellently.     

Including ligand‐induced protein flexibility into protein tunnel prediction

In proteins with buried active sites, understanding how ligands migrate through the tunnels that connect the exterior of the protein to the active site can shed light on substrate specificity and enzyme function. A growing body of evidence highlights the importance of protein flexibility in the binding site on ligand binding; however, the influence of protein flexibility throughout the body of the protein during ligand entry and egress is much less characterized. We have developed a novel tunnel prediction and evaluation method named IterTunnel, which includes the influence of ligand‐induced protein flexibility, guarantees ligand egress, and provides detailed free energy information as the ligand proceeds along the egress route. IterTunnel combines geometric tunnel prediction with steered molecular dynamics in an iterative process to identify tunnels that open as a result of ligand migration and calculates the potential of mean force of ligand egress through a given tunnel. Applying this new method to cytochrome P450 2B6, we demonstrate the influence of protein flexibility on the shape and accessibility of tunnels. More importantly, we demonstrate that the ligand itself, while traversing through a tunnel, can reshape tunnels due to its interaction with the protein. This process results in the exposure of new tunnels and the closure of preexisting tunnels as the ligand migrates from the active site. © 2014 Wiley Periodicals, Inc.     

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium ATPase is a member of the P‐type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X‐ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca²⁺ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca²⁺ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca²⁺ ion binds to site I , and then, the second Ca²⁺ ion approaches. The interactions between the second Ca²⁺ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca²⁺. Because of the electrostatic repulsion of the second Ca²⁺ and the electrostatic attraction of site II , the first bound Ca²⁺ shifts from site I to site II . Concertedly, the second Ca²⁺ binds to site I , forming a binding state with two Ca²⁺ ions, one at site I and the other at site II . Both of Glu908 and Asp800 coordinate with the two Ca²⁺ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Site Density Functional Theory and Structural Bioinformatics Analysis of the SARS-CoV Spike Protein and hACE2 Complex

The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by the SARS-CoV-2 spike (S) glycoprotein, which critically depends on the formation of complexes involving the spike protein receptor-binding domain (RBD) and the human cellular membrane receptor angiotensin-converting enzyme 2 (hACE2). Using classical site density functional theory (SDFT) and structural bioinformatics methods, we investigate binding and conformational properties of these complexes and study the overlooked role of water-mediated interactions. Analysis of the three-dimensional reference interaction site model (3DRISM) of SDFT indicates that water mediated interactions in the form of additional water bridges strongly increases the binding between SARS-CoV-2 spike protein and hACE2 compared to SARS-CoV-1-hACE2 complex. By analyzing structures of SARS-CoV-2 and SARS-CoV-1, we find that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) has expanded in size, indicating large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits stronger intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction causes a significant structural rigidification of the spike protein, favoring proteolytic processing of the S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification of the RBD dynamics and their impact on infectivity.     

Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism

According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.     

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

The nonheme iron enzyme OrfP reacts with l -Arg selectively to form the 3R,4R-dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l -Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C³−H hydrogen atom abstraction. This happens despite the fact that the C³−H and C⁴−H bond strengths of l -Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C³−H activation to a low-energy ⁵σ-pathway, while substrate positioning destabilizes the C⁴−H abstraction and sends it over the higher-lying ⁵π-pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.     

Preorganization and protein dynamics in enzyme catalysis

Recently, an alternative has been offered to the concept of transition state (TS) stabilization as an explanation for rate enhancements in enzyme-catalyzed reactions. Instead, most of the rate increase has been ascribed to preorganization of the enzyme active site to bind substrates in a geometry close to that of the TS, which then transit the activation barrier impelled by motions along the reaction coordinate. The question as to how an enzyme achieves such preorganization and concomitant TS stabilization as well as potential coupled motions along the reaction coordinate leads directly to the role of protein dynamic motion. Dihydrofolate reductase (DHFR) is a paradigm in which the role of dynamics in catalysis continues to be unraveled by a wealth of kinetic, structural, and computational studies. DHFR has flexible loop regions adjacent to the active site whose motions modulate passage through the kinetically preferred pathway. The participation of residues distant from the DHFR active site in enhancing the rate of hydride transfer, however, is unanticipated and may signify the importance of long range protein motions. The general significance of protein dynamics in understanding other biological processes is briefly discussed.     

Dissociative Adsorption of H2S on Li(110) Surface Using Density Functional Theory Calculations and Car-Parrinello Molecular Dynamics Simulations

The information concerning dissociative adsorption of H₂S on Li surface is inadequate and the mechanistic insight for its complete dissociation is yet to be explored. The present investigation aims to scrutinize the dissociative adsorption of H₂S on Li(110) surface using density functional theory calculations. The climbing image nudged elastic band calculation was employed to unveil the relative energy profiles for S−H dissociation. To elucidate the components of interaction energy responsible for stabilizing the adsorbed moieties on the surface, periodic energy decomposition analysis was performed. A Car-Parrinello molecular dynamics (CPMD) simulation was performed to understand the dynamic behaviour of H₂S on Li(110). Results vividly demonstrates: (i) partially dissociated product with perpendicular S−H is comparatively stable than the parallel SH, (ii) completely dissociated moieties H/H/S are the most stable among all, (iii) dissociation of first S−H is barrierless and the second S−H dissociation is a low energy barrier reaction, (iv) complete dissociation of H₂S occurs in a stepwise manner, (v) orbital and electrostatic contributions of the interaction energy plays a vital role in stabilizing the dissociated moieties, and (vi) stepwise dissociation of H₂S was further reinforced by CPMD.