Structure and function of malic enzymes, a new class of oxidative decarboxylases

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.     

Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic alpha-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (beta/alpha)7 barrel fold, and a C-terminal domain (domain II), which has a twisted beta-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu123 and Asp214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi alpha-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.     

Crystallographic and biophysical analysis of a bacterial signal peptidase in complex with a lipopeptide-based inhibitor

We report here the crystallographic and biophysical analysis of a soluble, catalytically active fragment of the Escherichia coli type I signal peptidase (SPase Delta2-75) in complex with arylomycin A2. The 2.5-A resolution structure revealed that the inhibitor is positioned with its COOH-terminal carboxylate oxygen (O45) within hydrogen bonding distance of all the functional groups in the catalytic center of the enzyme (Ser90 O-gamma, Lys145 N-zeta, and Ser88 O-gamma) and that it makes beta-sheet type interactions with the beta-strands that line each side of the binding site. Ligand binding studies, calorimetry, fluorescence spectroscopy, and stopped-flow kinetics were also used to analyze the binding mode of this unique non-covalently bound inhibitor. The crystal structure was solved in the space group P4(3)2(1)2. A detailed comparison is made to the previously published acyl-enzyme inhibitor complex structure (space group: P2(1)2(1)2) and the apo-enzyme structure (space group: P4(1)2(1)2). Together this work provides insights into the binding of pre-protein substrates to signal peptidase and will prove helpful in the development of novel antibiotics.     

A catalytic domain exosite (Cys527-Cys542) in factor XIa mediates binding to a site on activated platelets

The zymogen, factor XI, and the enzyme, factor XIa, interact specifically with functional receptors on the surface of activated platelets. These studies were initiated to identify the molecular subdomain within factor XIa that binds to activated platelets. Both factor XIa (Ki approximately 1.4 nM) and a chimeric factor XIa containing the Apple 3 domain of prekallikrein (Ki approximately 2.7 nM) competed with [125I]factor XIa for binding sites on activated platelets, suggesting that the factor XIa binding site for platelets is not located in the Apple 3 domain which mediates factor XI binding to platelets. The recombinant catalytic domain (Ile370-Val607) inhibited the binding of [125I]factor XIa to the platelets (Ki approximately 3.5 nM), whereas the recombinant factor XI heavy chain did not, demonstrating that the platelet binding site is located in the light chain of factor XIa. A conformationally constrained cyclic peptide (Cys527-Cys542) containing a high-affinity (KD approximately 86 nM) heparin-binding site within the catalytic domain of factor XIa also displaced [125I]factor XIa from the surface of activated platelets (Ki approximately 5.8 nM), whereas a scrambled peptide of identical composition was without effect, suggesting that the binding site in factor XIa that interacts with the platelet surface resides in the catalytic domain near the heparin binding site of factor XIa. These data support the conclusion that a conformational transition accompanies conversion of factor XI to factor XIa that conceals the Apple 3 domain factor XI (zymogen) platelet binding site and exposes the factor XIa (enzyme) platelet binding site within the catalytic domain possibly comprising residues Cys527-Cys542.     

Two crystal forms of helix II of Xenopus laevis 5S rRNA with a cytosine bulge

The crystal structure of r(GCCACCCUG).r(CAGGGUCGGC), helix II of the Xenopus laevis 5S rRNA with a cytosine bulge (underlined), has been determined in two forms at 2.2 A (Form I, space group P4(2)2(1)2, a = b = 57.15 A and c = 43.54 A) and 1.7 A (Form II, space group P4(3)2(1)2, a = b = 32.78 A and c = 102.5 A). The helical regions of the nonamers are found in the standard A-RNA conformations and the two forms have an RMS deviation of 0.75 A. However, the cytosine bulge adopts two significantly different conformations with an RMS deviation of 3.9 A. In Form I, the cytosine bulge forms an intermolecular C+*G.C triple in the major groove of a symmetry-related duplex with intermolecular hydrogen bonds between N4C and O6G, and between protonated N3+C and N7G. In contrast, a minor groove C*G.C triple is formed in Form II with intermolecular hydrogen bonds between O2C and N2G, and between N3C and N3G with a water bridge. A partial major groove opening was observed in Form I structure at the bulge site. Two Ca2+ ions were found in Form I helix whereas there were none in Form II. The structural comparison of these two forms indicates that bulged residues can adopt a variety of conformations with little perturbation to the global helix structure. This suggests that bulged residues could function as flexible latches in bridging double helical motifs and facilitate the folding of large RNA molecules.     

Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies

During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.     

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.     

Structural basis of inhibition revealed by a 1:2 complex of the two-headed tomato inhibitor-II and subtilisin Carlsberg

Multidomain proteinase inhibitors play critical roles in the defense of plants against predation by a wide range of pests. Despite a wealth of structural information on proteinase-single domain inhibitor interactions, the structural basis of inhibition by multidomain proteinase inhibitors remains poorly understood. Here we report the 2.5-A resolution crystal structure of the two-headed tomato inhibitor-II (TI-II) in complex with two molecules of subtilisin Carlsberg; it reveals how a multidomain inhibitor from the Potato II family of proteinase inhibitors can bind to and simultaneously inhibit two enzyme molecules within a single ternary complex. The N terminus of TI-II initiates the folding of Domain I (Lys-1 to Cys-15 and Pro-84 to Met-123) and then completes Domain II (Ile-26 to Pro-74) before coming back to complete the rest of Domain I (Pro-84 to Met-123). The two domains of TI-II adopt a similar fold and are arranged in an extended configuration that presents two reactive site loops at the opposite ends of the inhibitor molecule. Each subtilisin molecule interacts with a reactive site loop of TI-II through the standard, canonical binding mode. Remarkably, a significant distortion of the active site of subtilisin is induced by the presence of phenylalanine in the P1 position of reactive site loop II of TI-II. The structure of the TI-II.(subtilisin)2 complex provides a molecular framework for understanding how multiple inhibitory domains in a single Potato II type proteinase inhibitor molecule from the Potato II family act to inhibit proteolytic enzymes.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

Probing the Ubiquinone Reduction Site in Bovine Mitochondrial Complex I Using a Series of Synthetic Ubiquinones and Inhibitors

Studies of the structure–activity relationships of ubiquinones and specific inhibitors are helpful to probe the structural and functional features of the ubiquinone reduction site of bovine heart mitochondrial complex I. Bulky exogenous short-chain ubiquinones serve as sufficient electron acceptors from the physiological ubiquinone reduction site of bovine complex I. This feature is in marked contrast to other respiratory enzymes such as mitochondrial complexes II and III. For various complex I inhibitors, including the most potent inhibitors, acetogenins, the essential structural factors that markedly affect the inhibitory potency are not necessarily obvious. Thus, the loose recognition by the enzyme of substrate and inhibitor structures may reflect the large cavitylike structure of the ubiquinone (or inhibitor) binding domain in the enzyme. On the other hand, several phenomena are difficult to explain by a simple one-catalytic site model for ubiquinone.     

Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

Sulphamate and sulphamide derivatives have been largely investigated as carbonic anhydrase inhibitors (CAIs) by means of different experimental techniques. However, the structural determinants responsible for their different binding mode to the enzyme active site were not clearly defined so far. In this paper, we report the X-ray crystal structure of hCA II in complex with a sulphamate inhibitor incorporating a nitroimidazole moiety. The comparison with the structure of hCA II in complex with its sulphamide analogue revealed that the two inhibitors adopt a completely different binding mode within the hCA II active site. Starting from these results, we performed a theoretical study on sulphamate and sulphamide derivatives, demonstrating that electrostatic interactions with residues within the enzyme active site play a key role in determining their binding conformation. These findings open new perspectives in the design of effective CAIs using the sulphamate and sulphamide zinc binding groups as lead compounds.     

Structure of Golgi alpha-mannosidase II: a target for inhibition of growth and metastasis of cancer cells.

Golgi alpha-mannosidase II, a key enzyme in N-glycan processing, is a target in the development of anti- cancer therapies. The crystal structure of Drosophila Golgi alpha-mannosidase II in the absence and presence of the anti-cancer agent swainsonine and the inhibitor deoxymannojirimycin reveals a novel protein fold with an active site zinc intricately involved both in the substrate specificity of the enzyme and directly in the catalytic mechanism. Identification of a putative GlcNAc binding pocket in the vicinity of the active site cavity provides a model for the binding of the GlcNAcMan(5)GlcNAc(2) substrate and the consecutive hydrolysis of the alpha1,6- and alpha1,3-linked mannose residues. The enzyme-inhibitor interactions observed provide insight into the catalytic mechanism, opening the door to the design of novel inhibitors of alpha-mannosidase II.     

Carbonic anhydrase inhibitors: Valdecoxib binds to a different active site region of the human isoform II as compared to the structurally related cyclooxygenase II "selective" inhibitor celecoxib

The high resolution X-ray crystal structure of the adduct of human carbonic anhydrase (CA, EC 4.2.1.1) isoform II (hCA II) with the clinically used painkiller valdecoxib, acting as a potent CA II and cyclooxygenase-2 (COX-2) inhibitor, is reported. The ionized sulfonamide moiety of valdecoxib is coordinated to the catalytic Zn(II) ion with a tetrahedral geometry. The phenyl-isoxazole moiety of the inhibitor fills the active site channel and interacts with the side chains of Gln92, Val121, Leu198, Thr200, and Pro202. Its 3-phenyl group is located into a hydrophobic pocket, simultaneously establishing van der Waals interactions with the aliphatic side chain of various hydrophobic residues (Val135, Ile91, Val121, Leu198, and Leu141) and a strong offset face-to-face stacking interaction with the aromatic ring of Phe131 (the chi1 angle of which is rotated about 90 degrees with respect to what was observed in the structure of the native enzyme and those of other sulfonamide complexes). Celecoxib, a structurally related COX-2 inhibitor for which the X-ray crystal structure was reported earlier, binds in a completely different manner to hCA II as compared to valdecoxib. Celecoxib completely fills the entire CA II active site, with its trifluoromethyl group in the hydrophobic part of the active site and the p-tolyl moiety in the hydrophilic one, not establishing any interaction with Phe131. In contrast to celecoxib, valdecoxib was rotated about 90 degrees around the chemical bond connecting the benzensulfonamide and the substituted isoxazole ring allowing for these multiple favorable interactions. These different binding modes allow for the further drug design of various CA inhibitors belonging to the benzenesulfonamide class.     

Crystal structure of a statin bound to a class II hydroxymethylglutaryl-CoA reductase

Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the primary target in the current clinical treatment of hypercholesterolemias with specific inhibitors of the "statin" family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzymes of important bacterial pathogens. To investigate the molecular basis for this difference we determined the x-ray structure of the class II Pseudomonas mevalonii HMG-CoA reductase in complex with the statin drug lovastatin. The structure shows lovastatin bound in the active site and its interactions with residues critically involved in catalysis and substrate binding. Binding of lovastatin also displaces the flap domain of the enzyme, which contains the catalytic residue His-381. Comparison with the structures of statins bound to the human enzyme revealed a similar mode of binding but marked differences in specific interactions that account for the observed differences in affinity. We suggest that these differences might be exploited to develop selective class II inhibitors for use as antibacterial agents against pathogenic microorganisms.     

Evidence for a novel binding site conformer of aldose reductase in ligand-bound state

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of how the slight change of a chemically extended side-chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships.     

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.     

Homology modeling and docking mechanism of the mercaptosuccinate and methotrexate to P. falciparum 1-Cys peroxiredoxin: a preliminary molecular study

A three-dimensional (3-D) model of 1-Cys peroxiredoxin from P. falciparum (Pf-Prx) has been constructed by homology modeling. The model building was based on a structural alignment with the human 1-Cys peroxiredoxin ray structure. First, mercaptosuccinate was docked by Molecular and Quantum Mechanics at the active site in both isozymes, evidencing the role of different residues in the ligand-protein interaction. Stable conformation of the inhibitor in the active site was obtained from the conformational analysis by molecular dynamics. Next, The complex was reoptimized by semiempirical molecular orbital AM1 method. Conformational and frontier orbitals analyses of the ligand-protein complex were carried out in an attempt to obtain structural insight into the inhibition mechanism. Finally, the docking study of the methotrexate (MTX), an anticancer drug also used as an antimalarial inhibitor, into the modes binding site was performed. From the resulting stable complex structure, it was found that the glutamate ring of MTX fits the active site with high complementarity. The glutamate ring formed two hydrogen bonds to the imidazol group of His41 and the amino groups of Arg129. The side-chain of glutamate was in close proximity to the sulfur atom of the catalytic residue, Cys47. This binding mode suggests a possible inhibition mechanism, whereby the cysteine residue is covered with the glutamate ring of the MTX inhibitor, forming an enzyme-ligand adduct. In addition, the higher interaction energies and the molecular orbitals localization between the Pf-Prx active site and the inhibitors alluded to the probable binding sites of the ligand nucleophilic ring.