Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis.

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics simulations with small-angle X-ray scattering, mutagenesis, and heterodimer generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric interdomain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

Tandem Condensation-Cycloaddition of Propargylic Amines with α-Azido Ketones and ß-Alkoxy-γ-Azido Enones.

α-Azido ketones and their vinylogous relatives ß-alkoxy-γ-azido enones are versatile building blocks for constructing diverse heterocyclic products, but are prone to azide decomposition. Here, we report their condensation with propargylic amines and investigate the fate of the intermediate azido-enamine condensation products, both experimentally and theoretically. Efficient intramolecular cycloaddition was observed for electron-poor azide substrates, and a range of diversely substituted [1,2,3]triazolo[1,5-a]pyrazine products is reported. For electron-rich substrates, azide decomposition predominated. Computational modeling of possible pathways from the azido-enamine intermediates revealed two alternative mechanisms for azide decomposition, which were consistent with observed side products.

Precise Positioning of Water Is Critical for Hydrolysis Catalyzed by 5'-Methylthioadenosine Nucleosidase.

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (DN*AN) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported. To understand how subtle changes in sequence and structure give rise to differences in chemistry between homologous enzymes, we have probed the reaction coordinates of two MTAN enzymes using quantum mechanical/molecular mechanical and molecular dynamics simulations combined with experimental methods. We show that the transition state structure and energy are significantly affected by the recruitment and positioning of the catalytic water molecule and that subtle differences in the noncatalytic active site residues alter the environment of the catalytic water, leading to changes in the reaction coordinate and observed reaction rate.

The design of protozoan phosphoribosyltransferase inhibitors containing non-charged phosphate mimic residues.

Phosphate groups play essential roles in biological processes, including retention inside biological membranes. Phosphodiesters link nucleic acids, and the reversible transfer of phosphate groups is essential in energy metabolism and cell-signalling processes. Phosphorylated metabolic intermediates are known targets for metabolic and disease-related disorders, and the enzymes involved in these pathways recognize phosphate groups in their catalytic sites. Therapeutics that target these enzymes can require charged (ionic) entities to capture the binding energy of ionic substrates. Such compounds are not cell-permeable and require pro-drug strategies for efficacy as therapeutics. Protozoan parasites such as Plasmodium and Trypanosoma spp. are unable to synthesise purines de novo and rely on the salvage of purines from the host cell to synthesise free purine bases. Purine phosphoribosyltransfereases (PPRTases) play a crucial role for purine salvage and are potential target for drug development. Here we present attempts to design inhibitors of PPRTases that are non-ionic and show affinity for the nucleotide 5'-phosphate binding site. Inhibitor design was based on known potent ionic inhibitors, reported phosphate mimics and computational modelling studies.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Computational investigations of allostery in aromatic amino acid biosynthetic enzymes.

Allostery, in which binding of ligands to remote sites causes a functional change in the active sites, is a fascinating phenomenon observed in enzymes. Allostery can occur either with or without significant conformational changes in the enzymes, and the molecular basis of its mechanism can be difficult to decipher using only experimental techniques. Computational tools for analyzing enzyme sequences, structures, and dynamics can provide insights into the allosteric mechanism at the atomic level. Combining computational and experimental methods offers a powerful strategy for the study of enzyme allostery. The aromatic amino acid biosynthesis pathway is essential in microorganisms and plants. Multiple enzymes involved in this pathway are sensitive to feedback regulation by pathway end products and are known to use allostery to control their activities. To date, four enzymes in the aromatic amino acid biosynthesis pathway have been computationally investigated for their allosteric mechanisms, including 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, anthranilate synthase, chorismate mutase, and tryptophan synthase. Here we review the computational studies and findings on the allosteric mechanisms of these four enzymes. Results from these studies demonstrate the capability of computational tools and encourage future computational investigations of allostery in other enzymes of this pathway.

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis.

Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution. ATP-phosphoribosyltransferase (ATP-PRT), as the first enzyme in the histidine biosynthetic pathway, is allosterically regulated by histidine and offers a good platform for the study of allostery. Two forms of ATP-PRT, namely long and short forms, were discovered that show different arrangements of their regulatory machinery. Crystal structures of the long-form ATP-PRT have revealed overall conformational changes in the inhibited state, but the observed changes in the active state are quite subtle, making the elucidation of its allosteric mechanism difficult. Here, we combine computational methods (ligand docking, quantum mechanics/molecular mechanics optimization, and molecular dynamic simulations) with experimental studies to probe the signal transmission between remote allosteric and active sites. Our results reveal that distinct conformational ensembles of the catalytic domain with different dynamic properties exist in the ligand-free and histidine-bound enzymes. These ensembles display different capabilities in supporting the catalytic and allosteric function of ATP-PRT. The findings give insight into the underlying mechanism of allostery and allow us to propose that the hinge twisting within the catalytic domain is the key for both enhancement of catalysis and provision of regulation in ATP-PRT enzymes.

A dimeric catalytic core relates the short and long forms of ATP-phosphoribosyltransferase.

Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an allosteric inhibitor. Here, we describe a truncated long-form ATP-PRT from Campylobacter jejuni devoid of its regulatory domain (CjeATP-PRTcore). Results showed that CjeATP-PRTcore is dimeric, exhibits attenuated catalytic activity, and is insensitive to histidine, indicating that the covalently linked regulatory domain plays a role in both catalysis and regulation. Crystal structures were obtained for CjeATP-PRTcore in complex with both substrates, and for the first time, the complete product of the reaction. These structures reveal the key features of the active site and provide insights into how substrates move into position during catalysis.

Structure of the NDH-2 - HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors.

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1-UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.

'Tethering' fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110 µM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.

Probing the determinants of phosphorylated sugar-substrate binding for human sialic acid synthase.

N-acetylneuraminic acid (NeuNAc), the most naturally abundant sialic acid, is incorporated as the terminal residue of mammalian cell surface glycoconjugates and acts as a key facilitator of cellular recognition, adhesion and signalling. Several pathogenic bacteria similarly express NeuNAc on their cell surfaces, allowing evasion of their host's immune system. Prokaryotic NeuNAc biosynthesis proceeds via condensation of phosphoenolpyruvate (PEP) with N-acetylmannosamine (ManNAc), a reaction catalysed by the domain-swapped homodimeric enzyme, N-acetylneuraminic acid synthase (NeuNAcS). Conversely, the mammalian orthologue, N-acetylneuraminic acid 9-phosphate synthase (NeuNAc 9-PS) utilises the phosphorylated substrate N-acetylmannosamine 6-phosphate (ManNAc 6-P) in catalysis. Here we report an investigation into the determinants of substrate specificity of human NeuNAc 9-PS, using model-guided mutagenesis to delineate binding interactions with ManNAc 6-P. Modelling predicts the formation of a domain-swapped homodimer as observed for bacterial variants, which was supported by experimental small angle X-ray scattering. A number of conserved residues which may play key roles in the selection of ManNAc 6-P were identified and substituted for alanine to assess their function. Lys290 and Thr80 were identified as a putative phosphate binding pair, with the cationic lysine residue extending into the active site from the adjacent chain of the dimeric enzyme. Substitution of these residues results in a significant loss of activity and reduced affinity for ManNAc 6-P. These residues, along with the electropositive ß2α2 loop, are likely to facilitate the PEP dependent binding and stabilisation of ManNAc 6-P. By utilising a phosphorylated sugar-substrate, the mammalian enzyme gains considerable catalytic affinity advantage over its bacterial counterpart.

Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase.

N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N-terminal catalytic (ß/α)8 barrel linked to a C-terminal antifreeze protein-like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X-ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen-bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries.

An efficient regioconvergent synthesis of 3-aza-obeticholic acid.

Bile acids (BAs) are steroidal molecules that play important roles in nutrient absorption, distribution, and excretion. They also act on specific receptors implicated in various metabolic and inflammatory diseases demonstrating their importance as potential drug candidates. Accordingly, there has been a concerted effort to develop new BA derivatives to probe structure-activity relationships with the goal of discovering BA analogues with enhanced pharmacological properties. Among the many steroidal derivatisations reported, the formation of endocyclic azasteroids appeals due to their potential to deliver altered biological responses with minimal change to the steroidal superstructure. Here, we report the synthesis of 3-aza-obeticholic acid (6) via a regioconvergent route. Ammoniolysis of lactones, formed from an m-CPBA-mediated Baeyer-Villiger reaction on a 3-keto-OCA derivative, furnished protected intermediate amido-alcohols which were separately elaborated to amino-alcohols via Hofmann degradation with BAIB. Upon individual N-Boc-protection, these underwent annulation to the 3-aza-A-ring when subjected to either mesylation or a Dess-Martin oxidation/hydrogenation sequence. Global deprotection of the 3-aza-intermediate delivered 3-aza-OCA in ten steps and an overall yield of up to 19%.

Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Arg314 is essential for catalysis by N-acetyl neuraminic acid synthase from Neisseria meningitidis.

The sialic acid N-acetylneuraminic acid (NANA) has a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host's immune system. NANA is synthesized in bacteria by the homodimeric enzyme NANA synthase (NANAS), which catalyzes a condensation reaction between phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc). NANAS is closely related to the α-keto acid synthases 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase and 3-deoxy-d-manno-octulosonate 8-phosphate synthase. NANAS differs from these enzymes in that it contains an antifreeze protein like (AFPL) domain, which extends from the C-terminal of the (ß/α)8 barrel containing the active site and contributes a highly conserved arginine (Arg314) into the active site of the opposing monomer chain. We have investigated the role of Arg314 in NmeNANAS through mutagenesis and a combination of kinetic and structural analyses. Using isothermal titration calorimetry and molecular modeling, we have shown that Arg314 is required for the catalytic function of NANAS and that the delocalized positively charged guanidinium functionality of this residue provides steering of the sugar substrate ManNAc for suitable placement in the active site and thus reaction with PEP.

Structure, mechanism and inhibition of anthranilate phosphoribosyltransferase.

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho-d-ribose 1-diphosphate (PRPP) binds to the C-terminal domain and coordinates to Mg2+, in a site completed by two flexible loops. Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Engineering broad-spectrum inhibitors of inflammatory chemokines from subclass A3 tick evasins.

Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from "classical" class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.

The discovery of 12ß-methyl-17-epi-18-nor-bile acids as potent and selective TGR5 agonists.

Recent discoveries have demonstrated that the physiological function of bile acids extends to the regulation of diverse signaling processes through interactions with nuclear and G protein-coupled receptors, most notably the Farnesoid-X nuclear receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5). Targeting such signaling pathways pharmacologically, i.e. with bile acid-derived therapeutics, presents great potential for the treatment of various metabolic, inflammatory immune, liver, and neurodegenerative diseases. Here we report the discovery of two potent and selective TGR5 agonists (NZP196 and 917). These compounds are the taurine conjugates of 6α-ethyl-substituted 12ß-methyl-18-nor-bile acids with the side chain being located on the α-face of the steroid scaffold. The compounds emerged from a screening effort of a diverse library of 12ß-methyl-18-nor-bile acids that were synthesized from 12ß-methyl-18-nor-chenodeoxycholic acid and its C17-epimer. Upon testing for FXR activity, both compounds were found to be inactive, thus revealing selectivity for TGR5.

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: a feedback inhibition mechanism of the menaquinone biosynthesis pathway.

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus (Sau), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau-MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau-MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau, an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb-MenD and Sau-MenD, feedback inhibition by DHNA is a conserved mechanism in Sau-MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: combining mechanism- and modeling-based design.

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-D-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.