A guanidinium-based inhibitor of a type I isopentenyl diphosphate isomerase.

An inhibitor bearing a phosphinylphosphonate group appended to a guanidinium functionality was designed to inhibit enzymes that generate carbocations from dimethylallyl diphosphate. When tested against human farnesyl diphosphate synthase the inhibitor bound with high micromolar affinity and did not bind more tightly than an isosteric inhibitor lacking the guanidinium functionality. When tested against the Type I isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Escherichia coli, the inhibitor bound with a Ki value of 120 nM, which was 400 times greater than its isosteric counterpart. This strategy of inhibition was much more effective with an enzyme that generates a carbocation that is not stabilized by both resonance and ion pairing, presumably because there is more evolutionary pressure on the enzyme to stabilize the cation.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Type II isopentenyl diphosphate isomerase: irreversible inactivation by covalent modification of flavin.

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.

Synthesis and evaluation of substrate analogues as mechanism-based inhibitors of type II isopentenyl diphosphate isomerase.

Type 2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, contains a tightly bound molecule of FMN. To probe the mechanism of the reaction, cyclopropyl and epoxy substrate analogues, designed to be mechanism-based irreversible inhibitors, were synthesized and evaluated with IDI-2 from Thermus thermophilus. The cyclopropyl analogues were alternative substrates. The epoxy analogue was an irreversible inhibitor, with kI = 0.37 +/- 0.07 min(-1) and KI = 1.4 +/- 0.3 microM. LC-MS studies revealed formation of an epoxide-FMN adduct.

Metabolic mechanisms in heart failure.

Although neurohumoral antagonism has successfully reduced heart failure morbidity and mortality, the residual disability and death rate remains unacceptably high. Though abnormalities of myocardial metabolism are associated with heart failure, recent data suggest that heart failure may itself promote metabolic changes such as insulin resistance, in part through neurohumoral activation. A detrimental self-perpetuating cycle (heart failure --> altered metabolism --> heart failure) that promotes the progression of heart failure may thus be postulated. Accordingly, we review the cellular mechanisms and pathophysiology of altered metabolism and insulin resistance in heart failure. It is hypothesized that the ensuing detrimental myocardial energetic perturbations result from neurohumoral activation, increased adverse free fatty acid metabolism, decreased protective glucose metabolism, and in some cases insulin resistance. The result is depletion of myocardial ATP, phosphocreatine, and creatine kinase with decreased efficiency of mechanical work. On the basis of the mechanisms outlined, appropriate therapies to mitigate aberrant metabolism include intense neurohumoral antagonism, limitation of diuretics, correction of hypokalemia, exercise, and diet. We also discuss more novel mechanistic-based therapies to ameliorate metabolism and insulin resistance in heart failure. For example, metabolic modulators may optimize myocardial substrate utilization to improve cardiac function and exercise performance beyond standard care. The ultimate success of metabolic-based therapy will be manifest by its capacity further to lessen the residual mortality in heart failure.

Escherichia coli type 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.

Inhibition of type 2 isopentenyl diphosphate isomerase from Methanocaldococcus jannaschii by a mechanism-based inhibitor of type 1 isopentenyl diphosphate isomerase.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2, EC 5.3.3.2) is a flavoprotein, which requires FMN, NADPH, and Mg2+ for the activity to convert isopentenyl diphosphate to dimethylallyl diphosphate. For investigation of the reaction mechanism of IDI-2, 3,4-epoxy-3-methylbutyl diphosphate (EIPP), a mechanism-based inhibitor of type 1 IDI (IDI-1), was treated with the overexpressed IDI-2 (MjIDI) from methanogenic archaeon Methanocaldococcus jannaschii. EIPP showed the time- and concentration-dependent inhibition (KI; 56.5 mM, k(inact); 0.10 s(-1), k(inact)/KI; 1.76 s(-1)M(-1)) and the UV-vis spectrum of MjIDI after treatment with EIPP was apparently different from that of the untreated MjIDI. These results indicated that EIPP modified FMN through a covalent bond in the active site of MjIDI. The formed EIPP-FMN complex was separated from the reaction mixture and the spectrometric analysis of the complex suggested that the reduced form of FMN bound to EIPP at the N5 position. These results may suggest that the IDI-2 reaction is similar to IDI-1, which proceeds via carbocation-type intermediate.

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate.

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.

Supplementation with alpha-linolenic acid-rich diacylglycerol suppresses fatty liver formation accompanied by an up-regulation of beta-oxidation in Zucker fatty rats.

Insulin resistance-related obesity and diabetes mellitus are the predominant causes of fatty liver disease. Here we examine the effects of dietary diacylglycerol (DG), which is a minor component of plant oils, on lipid accumulation and the expression of genes involved in lipid metabolism in the liver. The animals were fed diets containing either 10% triacylglycerol (TG), 10% TG + 4% alpha-linolenic acid-rich TG (ALATG) or 10% TG + 4% alpha-linolenic acid-rich diacylglycerol (ALADG) for a period of 1 month. Supplementation with ALADG significantly inhibited hepatic triglyceride accumulation; this was accompanied by the up-regulation of beta-oxidation activity, and acyl-CoA oxidase (ACO) and medium-chain acyl-CoA dehydrogenase (MCAD) mRNA levels. By contrast, no significant changes were observed in the levels of peroxisome proliferator-activated receptor-alpha (PPARalpha) and sterol regulatory element-binding protein-1 (SREBP-1) mRNAs. These results indicate that ALADG might be useful in the prevention of fatty liver formation; this effect could be closely related to the stimulation of lipid catabolism in the liver. In addition, our findings suggest that both acylglycerol structure (that is, the structural difference between TG and DG) and fatty-acid species affect the nutritional behaviour of dietary lipids.

Intrinsic isomerase activity of medium-chain acyl-CoA dehydrogenase.

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We found that the enzyme has intrinsic isomerase activity, which was confirmed using incubation followed with HPLC analysis. The isomerase activity of the enzyme was thoroughly characterized through studies of kinetics, substrate specificity, pH dependence, and enzyme inhibition. E376 mutants were constructed, and mutant enzymes were purified and characterized. It was shown that E376 is the catalytic residue for both dehydrogenase and isomerase activities of the enzyme. The isomerase activity of medium-chain acyl-CoA dehydrogenase is probably a spontaneous process driven by thermodynamic equilibrium with the formation of a conjugated structure after deprotonation of substrate alpha proton. The energy level of the transition state may be lowered by a stable dienolate intermediate, which gains further stabilization via charge transfer with the electron-deficient FAD cofactor of the enzyme. This raises the question as to whether the dehydrogenase might function as an isomerase in vivo in conditions in which the activity of the isomerase is decreased.

New fatty acid oxidation inhibitors with increased potency lacking adverse metabolic and electrophysiological properties.

New inhibitors of palmitoylCoA oxidation were synthesized based on a structurally novel lead, CVT-3501 (1). Investigation of structure-activity relationships was conducted with respect to potency of inhibition of cardiac mitochondrial palmitoylCoA oxidation and metabolic stability. Potent and metabolically stable analogues 33, 42, and 43 were evaluated in vitro for cytochrome P450 inhibition and potentially adverse electrophysiological effects. Compound 33 was also found to have favorable pharmacokinetic properties in rat.

Crystal structure of the C67A mutant of isopentenyl diphosphate isomerase complexed with a mechanism-based irreversible inhibitor.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate. Analysis of the 1.97 A crystal structure of the inactive C67A mutant of E. coli isopentenyl diphosphate:dimethylallyl diphosphate isomerase complexed with the mechanism-based inactivator 3,4-epoxy-3-methyl-1-butyl diphosphate is in agreement with an isomerization mechanism involving Glu 116, Tyr 104, and Cys 67. In particular, the results are consistent with a mechanism where Glu116 is involved in the protonation step and Cys67 in the elimination step.

Structure and mechanism of action of isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase.

We have obtained the three-dimensional X-ray crystallographic structure of a C67A mutant Escherichia coli isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase (EC 5.3.3.2) complexed with the bromohydrin of isopentenylpyrophosphate, at 1.93 A resolution. The overall backbone fold is very similar to that obtained previously for the wild-type enzyme in the presence of a divalent metal cation (Mn2+ or Mg2+). However, in the new structure, there are two metal binding sites, not just one. The first metal binding site is occupied by Mn2+, coordinated to three histidine and two glutamate residues, while the second is occupied by Mg2+, coordinated to two bromohydrin-ligand phosphate oxygens, the carbonyl oxygen of A67, a carboxyl oxygen of E87, and two water molecules. The C3 hydroxyl group of the bromohydrin inhibitor is involved in a short hydrogen bond to the carboxyl group of E116, one of the two Mn-bound glutamates. The structure obtained is consistent with a mechanism of action of the enzyme in which the carboxyl group of E116 protonates the double bond in isopentenylpyrophosphate, forming a carbocation, followed by removal of a C2 proton by the thiolate of C67, in the wild-type enzyme. The inhibition of the enzyme by a wide variety of other potent inhibitors is also readily explained on the basis of the bromohydrin inhibitor structure.

Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 A) of complexes of Escherichia coli IPP.DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction.

Identification of a bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.

We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC(50) values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase.

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates.

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [(14)C]mevalonate (MVA), [(14)C]IPP, and [(14)C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [(14)C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [(14)C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action.

Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.

Bisphosphonates, synthetic compounds which suppress bone resorption, are used in the treatment of skeletal disorders. Their mode of action and intracellular targets have not yet been identified. Recent evidence suggested that enzymes of the mevalonate pathway are the potential targets. In this study, we examined the effect of four potent nitrogen (N)-containing bisphosphonates, clodronate and NH2-olpadronate, an inactive analogue of olpadronate, on isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and protein geranylgeranyl transferase I activity. We found that all N-containing bisphosphonates inhibited isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity dose dependently with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo, whereas clodronate and NH2-olpadronate had no effect. Furthermore, none of the bisphosphonates tested affected geranylgeranyl pyrophosphate synthase or geranylgeranyl transferase I activity. Our study reveals for the first time the intracellular target of N-containing bisphosphonates and supports the view that all bisphosphonates do not share the same molecular mechanism of action.

Characterization of the role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitory factor.

The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit. The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-11. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pKa measured for the pH dependence of kinact/KI (5.7 +/- 0.2) and that measured for the pH dependence of the kcat/Km for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pKa of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolis, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The structural and mechanistic parallels place 4-OT, CHMI, and MIF in a superfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.