Potential Transition State Analogue Research Articles

Small-Molecule Allosteric Inhibitors of Human Aspartate Transcarbamoylase Suppress Proliferation of Bone Osteosarcoma Epithelial Cells.

Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 μM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

Binding of l-Anilino-8-Napthalenesulfonate to Free and Coformycin-Complexed Bovine Adenosine Deaminase

Abstract: We have determined the number of binding sites and average binding affinity of the nonpolar probe l-anilino-8- napthalenesulfonate to free bovine adenosine deaminase and to its complex with coformycin, a potent transition state analogue. Our results indicate an increase in average binding affinity accompanied by a reduction in number of sites for the coformycin-complexed enzyme. Interpretation ofthese results in terms of the relative stabilities ofthe two enzyme states and to the catalytic efficiency of enzymes is presented.

Hybrid QM/MM Free-Energy Evaluation of Drug-Resistant Mutational Effect on the Binding of an Inhibitor Indinavir to HIV-1 Protease.

A human immunodeficiency virus-1 (HIV-1) protease is a homodimeric aspartic protease essential for the replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analogues have been developed. However, serious drug-resistant mutants have emerged. For understanding the molecular mechanism of the drug resistance, an accurate examination of the impacts of the mutations on ligand binding and enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of indinavir, a potent transition state analogue inhibitor, to the wild-type protein and a V82T/I84V drug-resistant mutant of the HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free-energy optimization technique which combines a highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of the MM protein environment by long-time molecular dynamics simulations. Through the free-energy calculations of protonation states of catalytic groups at the binding pocket and of the ligand-binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug resistance through the direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

Inhibitor-Dependent Usage of the S1' Specificity Pocket of ER Aminopeptidase 2.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is an intracellular enzyme involved in the processing of antigenic peptides intended for presentation by major histocompatibility complex class I (MHCI) molecules. Because of its role in regulating immune responses, ERAP2 is an emerging pharmacological target. Phosphinic pseudopeptides are potent transition-state analogue inhibitors of ERAP2. Previous structure-activity studies have revealed a complex but ambiguous relationship between the occupation of putative specificity pockets and the inhibitor efficacy. To address these problems, we solved crystal structures of ERAP2 in complex with two phosphinic pseudotripeptide inhibitors. Both compounds are found in the catalytic site in a canonical orientation for transition-state analogues and utilize the S1 and S2' pockets in a similar fashion. Strikingly, their P1' side chains exhibit different orientations and make interactions with distinct shallow pockets near the ERAP2 active site. These structures suggest that S1' pocket usage in ERAP2 may be inhibitor-dependent and constitute useful starting templates for the further optimization of this class of compounds.

1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies.

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with Ki = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with Ki* = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the “isomerization” step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called “transition state analog”, followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer’s disease and protection of central AChE against organophosphorus compounds.

Transition-State Analogues of Campylobacter jejuni 5'-Methylthioadenosine Nucleosidase.

Campylobacter jejuni is a Gram-negative bacterium responsible for food-borne gastroenteritis and associated with Guillain-Barré, Reiter, and irritable bowel syndromes. Antibiotic resistance in C. jejuni is common, creating a need for antibiotics with novel mechanisms of action. Menaquinone biosynthesis in C. jejuni uses the rare futalosine pathway, where 5'-methylthioadenosine nucleosidase ( CjMTAN) is proposed to catalyze the essential hydrolysis of adenine from 6-amino-6-deoxyfutalosine to form dehypoxanthinylfutalosine, a menaquinone precursor. The substrate specificity of CjMTAN is demonstrated to include 6-amino-6-deoxyfutalosine, 5'-methylthioadenosine, S-adenosylhomocysteine, adenosine, and 5'-deoxyadenosine. These activities span the catalytic specificities for the role of bacterial MTANs in menaquinone synthesis, quorum sensing, and S-adenosylmethionine recycling. We determined inhibition constants for potential transition-state analogues of CjMTAN. The best of these compounds have picomolar dissociation constants and were slow-onset tight-binding inhibitors. The most potent CjMTAN transition-state analogue inhibitors inhibited C. jejuni growth in culture at low micromolar concentrations, similar to gentamicin. The crystal structure of apoenzyme C. jejuni MTAN was solved at 1.25 Å, and five CjMTAN complexes with transition-state analogues were solved at 1.42 to 1.95 Å resolution. Inhibitor binding induces a loop movement to create a closed catalytic site with Asp196 and Ile152 providing purine leaving group activation and Arg192 and Glu12 activating the water nucleophile. With inhibitors bound, the interactions of the 4'-alkylthio or 4'-alkyl groups of this inhibitor family differ from the Escherichia coli MTAN structure by altered protein interactions near the hydrophobic pocket that stabilizes 4'-substituents of transition-state analogues. These CjMTAN inhibitors have potential as specific antibiotic candidates against C. jejuni.

Design and synthesis of bicyclic acetals as Beta Secretase (BACE1) inhibitors

Taking advantage of the structural similarity between aspartic proteases, small-molecule peptidomimetic inhibitors that already showed activity towards Secreted Aspartic Protease 2 as anti-Candida agents and HIV protease inhibitors were exploited as potential BACE1 inhibitors. A focused library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was synthesized and assayed towards BACE1 enzyme, resulting in the identification of a thiolactam-containing hit compound possessing IC50 in the low micromolar range, and confirming the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Development of transition state analogue inhibitors for N-acetylglycosyltransferases bearing D-psicoor D-tagatofuranose scaffolds

AbstractNew potential transition state analogue inhibitors for N-acetylglucosyltransferases (GnTs) were synthesised. These compounds based on psico- and tagatofuranose (structure) scaffold contained a 2-thiophenyl-1-O-diethylphosphate moiety mimicking the proposed model of the transition state of the enzymatic reaction catalysed by N-acetylglucosyltransferases. The synthesised compounds as well as their precursors were fully characterised by NMR, optical rotation and mass techniques. Anomeric configuration of tagatofuranose derivatives was confirmed by X-ray crystallography. Two types of potential human glycosyltransferase (GnTs) inhibitors representing donor UDP-GlcNAc, assigned for biological assays on human GnTs, were prepared.

Identification of constrained peptidomimetic chemotypes as HIV protease inhibitors

Small-molecule peptidomimetic inhibitors that already showed activity towards Secreted aspartic protease 2 as anti-Candida agents are herein presented as candidate HIV protease inhibitors. A library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was screened towards HIV protease, resulting in the identification of hit compounds possessing IC50 in the sub-micromolar range, and showing the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Analogs of iso-azepinomycin as potential transition-state analog inhibitors of guanase: Synthesis, biochemical screening, and structure–activity correlations of various selectively substituted imidazo[4,5-e][1,4]diazepines

Guanase is an important enzyme of the purine salvage pathway of nucleic acid metabolism and its inhibition has beneficial implications in viral, bacterial, and cancer therapy. The work described herein is based on a hypothesis that azepinomycin, a heterocyclic natural product and a purported transition state analog inhibitor of guanase, does not represent the true transition state of the enzyme-catalyzed reaction as closely as does iso-azepinomycin, wherein the 6-hydroxy group of azepinomycin has been translocated to the 5-position. Based on this hypothesis, and assuming that iso-azepinomycin would bind to guanase at the same active site as azepinomycin, several analogs of iso-azepinomycin were designed and successfully synthesized in order to gain a preliminary understanding of the hydrophobic and hydrophilic sites surrounding the guanase binding site of the ligand. Specifically, the analogs were designed to explore the hydrophobic pockets, if any, in the vicinity of N1, N3, and N4 nitrogen atoms as well as O5 oxygen atom of iso-azepinomycin. Biochemical inhibition studies of these analogs were performed using a mammalian guanase. Our results indicate that (1) increasing the hydrophobicity near O5 results in a negative effect, (2) translocating the hydrophobicity from N3 to N1 also results in decreased inhibition, (3) increasing the hydrophobicity near N3 or N4 produces significant enhancement of inhibition, (4) increasing the hydrophobicity at either N3 or N4 with a simultaneous increase in hydrophobicity at O5 considerably diminishes any gain in inhibition made by solely enhancing hydrophobicity at N3 or N4, and (5) finally, increasing the hydrophilic character near N3 has also a deleterious effect on inhibition. The most potent compound in the series has a Ki value of 8.0±1.5μM against rabbit liver guanase.

Plasmin Substrate Binding Site Cooperativity Guides the Design of Potent Peptide Aldehyde Inhibitors

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

ChemInform Abstract: Synthesis of Polyhydroxylated Cyclohexenyl Sulfides and Sulfoxides. Evaluation of Their Inhibitory Activity on α- and β-D-Glucosidases.

Abstract Racemic polyhydroxylated sulfides and sulfoxides have been prepared as potential transition-state analogs of the glucoside hydrolysis reaction, through a reaction sequence involving transformations of a ketone group into thioacetal, followed by partial oxidation to sulfoxide then regioselective thermal elimination of the sulfoxide to vinyl sulfide. These sulfides with two, three or four hydroxyl groups have been oxidized to the corresponding diastereoisomeric sulfoxides. All compounds, summarily tested as inhibitors of α- and β-glucosidases, were found to be very weak inhibitors and thus their biological properties were not studied in depth. Curiously, however, their inhibitory properties, which are in the 10 mM range, do not really depend upon the number of hydroxyl groups or upon the presence of polar sulfoxide. In order to get more insights, 2-phenyl sulfoxide-3,4,5-trihydroxycyclohex-1-ene ( 9a ) was studied in more detail using Brewers yeast α-glucosidase and was shown to give a mixed-type inhibition with a high K i of 45 mM.

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes—A novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC 50 of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 µM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC 50s of 0.159, 0.26 and 0.73 µM, respectively. The IC 50 for dGuo in the presence of 1 µM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 µM. dGTP in human lymphocytes is elevated and a 3–5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 µM (predose levels < 0.004 µM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

gamma-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Abeta42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of gamma-secretase, invariably increase the Abeta42:Abeta40 ratio. However, the mechanism by which these mutations affect gamma-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Abeta42:Abeta40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active gamma-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Abeta42:Abeta40 ratios. These data suggest that perturbations to gamma-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Abeta42:Abeta40 ratio.

BCX-4208 (RO5092888), a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Is a Novel, Potent Orally Active Anti-T-Cell and B-Cell Agent.

The profound suppression of T-cell immunity seen in patients with an inherited PNP deficiency supports the potential application of inhibitors of this purine salvage enzyme in the therapy of T-cell malignancies and T-cell mediated autoimmune diseases. About thirty percent of PNP deficient patients also show evidence of B-cell dysfunction. BCX-4208 is a novel potent transition state analog inhibitor of PNP (IC50 ~ 0.0005 μM) and in the presence of 10 μM deoxyguanosine (dGuo), inhibits human lymphocyte proliferation induced by MLR, IL-2 or Con-A with IC50s of 0.159, 0.26 and 0.73 μM, respectively. The IC50 for dGuo in the same assays in the presence of 1 μM BCX-4208 ranges from 1–3 μM. Neither BCX-4208 alone nor dGuo alone inhibits proliferation of lymphocytes. In the presence of PNP inhibitor, dGuo is converted to dGMP and then to dGTP. Accumulation of dGTP results in the alteration of deoxynucleotide (dNTP) pools, causing death of cells via a mechanism characteristic of apoptosis. In vitro data demonstrates that following exposure to BCX-4208 and dGuo, dGTP in human lymphocytes is elevated and a 5–8 fold increase in dGTP results in 50% inhibition of lymphocyte proliferation. Flow cytometric analyses of human lymphocytes using annexin-V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis not only in T cells (CD3+), but also in B cells (CD20+; CD19+) (Table 1). BCX-4208 is orally bioavailable in mice, can achieve maximal inhibition of PNP, and elevates plasma dGuo levels to 3–5 μM (predose levels < 0.004 μM), which is similar to levels seen in PNP deficient patients and to levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of not only T-cell mediated diseases but also B-cell mediated diseases. BCX- 4208 is currently undergoing early clinical investigation in patients with psoriasis.Table 1.Cell subsets% Apoptotic cells mean ± SEM (n = 4–7)VehicleTreatment groupCD3+7.2 ± 0.918.3 ± 3.7*CD20+24.0 ± 3.641.8 ± 6.2*CD19+25.0 ± 3.151.6 ± 7.4**p ≤ 0.01 compared to vehicle

Novel Naphthalene-N-sulfonyl-d-glutamic Acid Derivatives as Inhibitors of MurD, a Key Peptidoglycan Biosynthesis Enzyme,

Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition state analogues, displayed IC50 values ranging from 80 to 600 microM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.

DFT and Docking Study of Potential Transition State Analogue Inhibitors of Glycosyltransferases

Conformational behavior of the [(2S,3R,4R,5S)-3,4,5-trihydroxy-2-(phenylsulfanyl)tetrahydrofuran-2-yl]methyl sulfate anion (2), which is the potential transition state (TS) analogue of the inverting glycosyltransferases, was studied by means of two-dimensional potential-energy maps, using a density functional theory method at the B3LYP/6-31+G* level. The maps revealed the presence of eight low-energy domains which were refined at the B3LYP/6-311++G** level and led to six conformers in vacuum. In aqueous solution, two conformers dominate at equilibrium. The preferred conformers superimpose well with the transition state structure, as determined previously for glycosyltransferase GnT-I. The conformations of 2 in the active site of glycosyltransferase GnT-I were obtained by docking methods. It was found that one of the two best docking poses mimics the binding mode of TS. These results suggest that the proposed TS mimics 2 have the potential to be used as a scaffold for the design of TS analogue inhibitors.

Structural and Functional Characterization of Enantiomeric Glutamic Acid Derivatives as Potential Transition State Analogue Inhibitors of MurD Ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP- N-acetylmuramoyl- l-alanine: d-glutamate ligase (MurD) catalyses the addition of d-glutamic acid to the cytoplasmic intermediate UDP- N-acetylmuramoyl- l-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, d-glutamic acid ( d-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the d-Glu or the l-Glu moiety. The binding modes of N-sulfonyl- d-Glu and N-sulfonyl- l-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.

Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei

Trypanosoma brucei (TB) cultured in rat blood, bovine serum, or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to 3 orders of magnitude different with cholesterol as the major sterol (>99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structures of these sterols reveal a nonconventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24 alkylation reaction, 25-azalanosterol (25-AL; inhibition constant Ki = 39 nM), was found to inhibit the growth of the procyclic and bloodstream forms at an IC(50) of approximately 1 microM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using the 25-AL-treated procyclic form, which, compared with control cultures, caused a change in cellular sterol content from ergostenols to cholesterol. However, growth of the bloodstream form disrupted by 25-AL was not rescued by cholesterol absorption from the host, suggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that the SMT can be a new enzyme target for drug design.

Escherichia coliType I Isopentenyl Diphosphate Isomerase: Structural and Catalytic Roles for Divalent Metals

Isopentenyl diphosphate isomerase (IDI) catalyzes the essential conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) in the mevalonate entry into the isoprenoid biosynthetic pathway. Two convergently evolved forms of IDI are known. Type I IDI, which is found in Eukarya and many Bacteria, catalyzes the isomerization of IPP and DMAPP by a protonation-deprotonation mechanism. The enzyme requires two divalent metal ions for activity. An X-ray structure of type I IDI from crystals soaked with (N,N-dimethylamino)-1-ethyl diphosphate (NIPP), a potent transition-state analogue for the carbocationic intermediate in the isomerization reaction, shows one of the metals in a His(3)Glu(2) hexacoordinate binding site, while the other forms a bridge between the diphosphate moiety of the substrate and the enzyme (Wouters, J.; et al. J. Biol. Chem. 2003, 278, 11903). Reconstitution of metal-free recombinant Escherichia coli type I IDI with several divalent metals-Mg(2+), Mn(2+), Zn(2+), Co(2+), Ni(2+), and Cd(2+)-generated active enzyme. Freshly purified IDI contained substoichiometric levels of a single metal ion, presumably bound in the hexacoordinate site. When NIPP was added to the disruption and purification buffers of enzyme, the purified protein contained 0.72 equiv of Mg(2+), 0.92 equiv of Zn(2+), and 0.10 equiv of Mn(2+). These results are consistent with a structure in which Mg(2+) facilitates diphosphate binding and Zn(2+) or Mn(2+) occupies the hexacoordinate site.