Enzyme Cavity Research Articles

Nanoscale enzyme inhibitors: Fullerenes inhibit carbonic anhydrase by occluding the active site entrance

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar—low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1 nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.

New Bisquaternary Isoquinolinium Inhibitors of Brain Cholinesterases - Synthesis and Anticholinesterase Activity

Abstract: Alzheimer´s disease (AD) is one of the most discussed diseases of present time. Because of the aging of our population it is high threat for future. Due to this, new drugs combating AD are still developed. In this study, twelve bisquaternary isoquinolinium de-rivatives differing in the linker between two heteroarenium rings were synthesized and tested for their potency to inhibit brain cho-linesterases. According to the obtained in vitro results, the anticholinesterase activity grew up with the length of the connection chain. Possible binding to the enzyme cavity was described using software Autodock. Moreover, LogP and molecular volume of prepared com-pounds were predicted. As resulted, the highest probability of synthesized compounds to penetrate blood-brain barrier could be expected for the compound with linker having eight methylenes. Keywords: Quinolinium, Quaternary compounds, Inhibitor, Cholinesterase, Alzheimer´s disease, Anticholinergic. INTRODUCTION At present, there is increasing number of people suffering from diseases connecting with the increasing age. Among them, Alz-heimer´s disease (AD) is well discussed by the scientific commu-nity. It belongs along with cancer, AIDS and cardiovascular dis-eases among the highest threats influencing the whole population. Although this disease is known already for more than one hundred years, there is no treatment able to stop its degenerative progress. Currently, there are just few AD treatments (Donepezil, Galan-tamine, Rivastigmine, Tacrine, Memantine) approved by FDA. All of them, except Memantine, are potent inhibitors of acetylcho-linesterase (AChE; EC 3.1.1.7). Although the cholinergic approach is able to have only one- or two-year benefit, economically, one or two more years of the productive life are very important [1,2]. Due to this reason, development of new AChE inhibitors as new AD-drugs candidates is still possible approach how to combat AD [3-5]. During last years, there were published several articles discuss-ing penetration of quaternary compounds into the blood-brain bar-rier (BBB) [6-8]. Thanks to this, also quaternary compounds with increased lipophility could be considered as an alternative for treatment of AD or at least as its supplement. Recently, there were published several articles describing quaternary compounds (expe-cially pyridinium compounds) as potential AD treatment [9-12]. In 2004, we found that more potent inhibitors of AChE than pyridi-nium salts seem to be quaternary compounds with conjugated aro-matic rings (quinolinium, isoquinolinium or acridinium) [13]. Thanks to the added aromatic rings, also their lipophilicity is higher if compared with pyridinium compounds. And with the lipophilic-ity, hand in hand, higher penetration through the BBB can be ex-pected. Because of the fact that affinity of bisquaternary compounds is higher than that of monoquaternary ones, in this article, we turned our attention to bisquaternary isoquinolinium compounds differing in the length of the connection chain between two isoquinolines.

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.

Substrate Binding Mechanism of HIV-1 Protease from Explicit-Solvent Atomistic Simulations

The binding mechanism of a peptide substrate (Thr-Ile-Met-Met-Gln-Arg, cleavage site p2-NC of the viral polyprotein) to wild-type HIV-1 protease has been investigated by 1.6 micros biased all-atom molecular dynamics simulations in explicit water. The configuration space has been explored biasing seven reaction coordinates by the bias-exchange metadynamics technique. The structure of the Michaelis complex is obtained starting from the substrate outside the enzyme within a backbone rmsd of 0.9 A. The calculated free energy of binding is -6 kcal/mol, and the kinetic constants for association and dissociation are 1.3 x 10(6) M(-1) s(-1) and 57 s(-1), respectively, consistent with experiments. In the main binding pathway, the flaps of the protease do not open sizably. The substrate slides inside the enzyme cavity from the tight lateral channel. This may contrast with the natural polyprotein substrate which is expected to bind by opening the flaps. Thus, mutations might influence differently the binding kinetics of peptidomimetic ligands and of the natural substrate.

Reviewing the Polyolefin Cyclization Reaction of the C35 Polyprene Catalyzed by Squalene‐Hopene Cyclase

A review of the polycyclization reaction of the C(35) polyprenoid by squalene-hopene cyclase: Surprisingly, our results completely disagree with a previous publication in which it was reported that a hexacyclic skeleton was constructed as the single product. In our work many tri- and tetracyclic scaffolds were isolated, but no penta- or hexacycles. The reasons for the different results and the mechanism of the polycyclization reaction are discussed (see figure).Squalene-hopene cyclase (SHC) catalyzes the polycyclization of squalene (C(30)) to the pentacyclic hopene with regio- and stereochemical specificity. In this study, we reviewed the polycyclization reaction of the C(35) polyprenoid catalyzed by SHC. Surprisingly, our results completely disagreed with a previous publication in which it was reported that a hexacyclic skeleton was constructed as the single product in 10 % yield (I. Abe, H. Tanaka, H. Noguchi, J. Am. Chem. Soc. 2002, 124, 14514-14515). Our experimental results showed that many tri- and tetracyclic products, up to 12, including novel carbocyclic cores, were generated in a high conversion ratio (97 %), but no detectable amounts of the penta- and hexacycle were produced. The mechanisms for the formation of the C(35) polyprene products isolated by us are discussed in this paper. The following four conformations were generated during the polycyclization cascade: chair-chair-boat, chair-chair-chair, chair-chair-chair-boat, and chair-chair-chair-chair. Larger amounts of the false intermediates with 13alpha-H (tricycle) and 17alpha-H (tetracycle) were produced compared with the true intermediates (13beta-H and 17beta-H), which indicates that the C(35) polyprene cannot fold correctly in the enzyme cavity due to the extra C(5) unit appended to squalene. This would have promoted the formation of the aborted cyclization products with tri- and tetracycles. In addition, the fact that no penta- or hexacyclic products were formed further indicates that SHC does not have sufficient space to accommodate the entire carbon framework of C(35).

Design of grafted copper complex in mesoporous silica in defined coordination, hydrophobicity and confinement states

A bio-inspired synthesis of a silica grafted polydentate copper(II) complex is developed following the structural concept of metalloproteins where well defined metal ion coordination state, hydrophobic environment and confined space are present. Mesostructured porous silica of MCM-41 type replaces the proteic matrix while the pore surface is engineered according to a molecular stencil patterning technique combining both partial hydrophobization and site isolation in order to mimic the enzymatic cavity. The overall five-step synthesis includes the sol–gel formation of the silica matrix followed by partial removal of the structure directing agent and, sequential surface chemical modifications. This new methodology is illustrated here using trimethylsilyl functions to dilute bromopropylsilyl tripod tethers that undergo, directly in the pores, in a subsequent step nucleophilic substitution by a tetradentate ligand N,N′-bis(2-pyridinylmethyl)ethane-1,2-diamine (L42). The metallation of the grafted ligand is obtained in the final step by merely contacting the solid with copper(II) chloride or triflate ethanolic solutions. Different techniques such as powder XRD, N2 adsorption–desorption, elemental analysis, IR, XPS, EPR and EXAFS were combined together with an emphasis on quantification to reach a quasi-molecular description at each functionalisation step of the internal surface of the materials.

Synthesis Strategies to Design Structures for Catalytic Applications

Abstract Two approaches to synthesize silicon-based catalytic structures that aim at capturing the properties and functionalities of natural enzymes are described in this brief review: unit-by-unit synthesis of macromolecular units and templating/imprinting synthesis of nano-cages. The unit-by-unit approach mimics the peptide synthesis method, offers atomic control of the structure, but is inefficient in synthesizing large structures such as nanocages. The templating/imprinting method is more suitable for nanocages at the sacrifice of atomic control, and the nanocages obtained are shown to possess properties exhibited by enzyme cavities.

Carbonic anhydrase inhibitors. Interaction of the antitumor sulfamate EMD 486019 with twelve mammalian carbonic anhydrase isoforms: Kinetic and X-ray crystallographic studies

The new antitumor sulfamate EMD 486019 was investigated for its interaction with twelve catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I – XIV. Similarly to 667-Coumate, a structurally related compound in phase II clinical trials as steroid sulfatase/CA inhibitor with potent antitumor properties, EMD 486019 acts as a strong inhibitor of isozymes CA II, VB, VII, IX, XII, and XIV ( K Is in the range of 13–19 nM) being less effective against other isozymes ( K Is in the range of 66–3600 nM against hCA I, IV, VA, VI, and mCA XIII, respectively). The complete inhibition profile of 667-Coumate against these mammalian CAs is also reported here for the first time. Comparing the X-ray crystal structures of the two adducts of CA II with EMD 486019 and 667-Coumate, distinct orientations of the bound sulfamates within the enzyme cavity were observed, which account for their distinct inhibition profiles. CA II/IX potent inhibitors belonging to the sulfamate class are thus valuable clinical candidates with potential for development as antitumor agents with a multifactorial mechanism of action.

MolAxis: Efficient and accurate identification of channels in macromolecules

Channels and cavities play important roles in macromolecular functions, serving as access/exit routes for substrates/products, cofactor and drug binding, catalytic sites, and ligand/protein. In addition, channels formed by transmembrane (TM) proteins serve as transporters and ion channels. MolAxis is a new sensitive and fast tool for the identification and classification of channels and cavities of various sizes and shapes in macromolecules. MolAxis constructs corridors, which are pathways that represent probable routes taken by small molecules passing through channels. The outer medial axis of the molecule is the collection of points that have more than one closest atom. It is composed of two-dimensional surface patches and can be seen as a skeleton of the complement of the molecule. We have implemented in MolAxis a novel algorithm that uses state-of-the-art computational geometry techniques to approximate and scan a useful subset of the outer medial axis, thereby reducing the dimension of the problem and consequently rendering the algorithm extremely efficient. MolAxis is designed to identify channels that connect buried cavities to the outside of macromolecules and to identify TM channels in proteins. We apply MolAxis to enzyme cavities and TM proteins. We further utilize MolAxis to monitor channel dimensions along Molecular Dynamics trajectories of a human Cytochrome P450. MolAxis constructs high quality corridors for snapshots at picosecond time-scale intervals substantiating the gating mechanism in the 2e substrate access channel. We compare our results with previous tools in terms of accuracy, performance and underlying theoretical guarantees of finding the desired pathways. MolAxis is available on line as a web-server and as a stand alone easy-to-use program (http://bioinfo3d.cs.tau.ac.il/MolAxis/).

Synthesis and Characterization of Water‐Soluble Zinc, Cobalt(II) and Copper(II) Complexes with a Neutral Tripodal N,N,N‐Ligand: Crystal Structures of [(κ3N‐4‐TIPOiPr)Co(H2O)(κ2O‐NO3)]NO3 and [(κ3N‐4‐TIPOiPr)Cu(H2O)(κO‐SO4)], 4‐TIPOiPr = tris(2‐isopropylimidazol‐4(5)‐yl)phosphane oxide

AbstractThe development of ligands bearing substituents to model a hydrophobic enzyme cavity and concurrently showing sufficient solubility in aqueous media seems somehow paradox. The zinc and cobalt(II) nitrato (4, 5) and copper(II) sulfato (6) complexes of the new tris(2‐isopropylimidazol‐4(5)‐yl)phosphane oxide ligand (4‐TIPOiPr, 2a) show exactly these properties; in addition, the structure of the CoII complex 5·MeOH·Et2O shows striking similarity to the active site of CoII‐substituted carbonic anhydrase. 2a and its metal complexes are stable towards hydrolysis which is in contrast to the known sensitivity of (imidazol‐2‐yl)phosphane oxides.

Molecular Analysis of the Benastatin Biosynthetic Pathway and Genetic Engineering of Altered Fatty Acid−Polyketide Hybrids

The entire gene locus encoding the biosynthesis of the potent glutathione-S-transferase inhibitors and apoptosis inducers benastatin A and B has been cloned and sequenced. The cluster identity was unequivocally proven by deletion of flanking regions and heterologous expression in S. albus and S. lividans. Inactivation and complementation experiments revealed that a KSIII component (BenQ) similar to FabH is crucial for providing and selecting the rare hexanoate PKS starter unit. In the absence of BenQ, several novel penta- and hexacyclic benastatin derivatives with antiproliferative activities are formed. In total, five new compounds were isolated and fully characterized, and the chemical analysis was confirmed by derivatization. The most intriguing observation is that the ben PKS can utilize typical straight and branched fatty acid synthase primers. If shorter straight-chain starters are utilized, the length of the polyketide backbone is increased, resulting in the formation of an extended, hexacyclic ring system reminiscent of proposed intermediates in the griseorhodin and fredericamycin pathways. Analysis and manipulation of the hybrid fatty acid polyketide pathway provides strong support for the hypothesis that the number of chain elongations is dependent on the total size of the polyketide chain that is accommodated in the PKS enzyme cavity. Our results also further substantiate the potential of metabolic engineering toward polyphenols with altered substituents and ring systems.

Probing the Acetylcholinesterase Inhibition of Sarin: A Comparative Interaction Study of the Inhibitor and Acetylcholine with a Model Enzyme Cavity

Interaction energies have been estimated between sarin and a model enzyme cavity of acetylcholinesterase (ACHE) using the density functional and Møller-Plesset second-order perturbation (MP2) levels of theories. The calculated interaction energies have been compared with those of acetylcholine and the same model ACHE cavity. The ACHE...sarin and ACHE...acetylcholine (Ach) structures have been optimized using DFT based two-layer ONIOM hybrid calculations. The nature of interactions has been investigated in detail using an interaction energy partitioning technique. The effects of solvation on the interaction energies have also been taken into account. An inhibition mechanism during the uptake of sarin inside the ACHE cavity has been proposed from the comparison of the energetics of the ACHE...sarin and ACHE...Ach complexes.

One-Pot and Sequential Organic Chemistry on an Enzyme Surface to Tether a Fluorescent Probe at the Proximity of the Active Site with Restoring Enzyme Activity

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Human Carbonic Anhydrase III: Structural and Kinetic Study of Catalysis and Proton Transfer,

The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.

Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB

Topoisomerases relieve the torsional strain in DNA that is built up during replication and transcription. They are vital for cell proliferation and are a target for poisoning by anti-cancer drugs. Type IB topoisomerase (TopIB) forms a protein clamp around the DNA duplex and creates a transient nick that permits removal of supercoils. Using real-time single-molecule observation, we show that TopIB releases supercoils by a swivel mechanism that involves friction between the rotating DNA and the enzyme cavity: that is, the DNA does not freely rotate. Unlike a nicking enzyme, TopIB does not release all the supercoils at once, but it typically does so in multiple steps. The number of supercoils removed per step follows an exponential distribution. The enzyme is found to be torque-sensitive, as the mean number of supercoils per step increases with the torque stored in the DNA. We propose a model for topoisomerization in which the torque drives the DNA rotation over a rugged periodic energy landscape in which the topoisomerase has a small but quantifiable probability to religate the DNA once per turn.

Experimental and theoretical approaches into the C- and D-ring problems of sterol biosynthesis. Hydride shift versus C–C bond migration due to cation conformational changes controlled by the counteranion

The C- and D-ring problems of sterol biosynthesis, how an enzyme overcomes the Markovnikov wall, were investigated by using a model compound from an experimental as well as theoretical standpoint. When model diol 20 was treated with BF 3·Et 2O, SnCl 4, TiF 4, Sc(OTf) 3, FeCl 3, or TfOH, spirocyclic ether 21 was formed as the sole product via a tert-cationic intermediate 16 through 1,2-hydride shift. However, the treatment with TiCl 4 afforded six-membered ring products 22 , 23 , 24 , 25 , 26 , and 27 via the ring expansion into the unstable six-membered ring secondary cation 17 . Occurrence of both α and β chloride 23 and 24 is distinctive evidence of the existence of secondary cation 17 , ruling out the idea of the concerted mechanism. Molecular mechanics calculations of the naked cation 15 elucidated two possible conformers, parallel 15- I (five membered ring and cationic plane) that is favorable for the hydride shift generating 16 and perpendicular 15- II leading to C–C bond migration to 17 . The first ab initio calculation of the cation conformation in the presence of counteranions such as [TiCl 4OH] −, [TiF 4OH] −, [BF 3OH] −, and [OTf] − entirely supported our experimental results. Although the counteranion [TiCl 4OH] − stabilizes perpendicular cation 15- II , it destabilizes the parallel conformer 15- I significantly, and thus, the C–C bond migration to 17 becomes the only possible pass. On the other hand, [TiF 4OH] −, [BF 3OH] −, and [OTf] − stabilize parallel conformer 15- I and the hydride shift to 16 becomes the only possible pass. The relative location or distance of the counteranion from the cation should be the biggest factor to control the stability and, thus, the conformation of the cation. Our results indicate that the carboxylate anions in the enzyme cavity enable to control the conformation of pre-C-ring cationic intermediate 3 to be perpendicular leading to six-membered C-ring secondary cation 4 . The parallel conformation of the cation 5 could lead to hydride shift to give tirucallanoids or lanostanoids. Therefore, this result is the first example that overcame the big Markovnikov wall experimentally and theoretically at least to our knowledge.

Active-site mutants of class B beta-lactamases: substrate binding and mechanistic study.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.

Enzymatic silicone oligomerization catalyzed by a lipid-coated lipase.

A lipid (1)-coated lipase can catalyze the oligomerization of diethoxydimethylsilane (DEDMS) in isooctane containing 2wt% water, where the polymerization occurs at the OH group of the coating lipid (1) in the enzyme cavity.

X-ray diffraction study of the complexes of SAICAR synthase with adenosinetriphosphate

The three-dimensional structures of two enzyme-substrate complexes of SAICAR synthase from the yeast Saccharomyces cerevisiae with adenosinetriphosphate (ATP) prepared under different conditions were studied by X-ray diffraction analysis and then refined. An enzyme molecule was shown to contain two binding sites of ATP. One of these sites is located in the central cavity of the enzyme molecule and apparently binds the ATP molecule directly involved in the enzymatic reaction. In the complexes, the phosphate groups of ATP occupying this site adopt different conformations depending on the Mg2+ concentration. The functional role of the second binding site located at a distance of approximately 15 A from the first site away from the central enzyme cavity has not been understood as yet. It might be that the second site perform the regulatory role in enzyme functioning.

Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III

The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP-protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of beta-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH-ACP interaction. Our experiments reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.