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At least 325 records · Page 18Linked to original sources
PubMed1987

Biosynthesis of the sesquiterpene patchoulol from farnesyl pyrophosphate in leaf extracts of Pogostemon cablin (patchouli): mechanistic considerations.

Several mechanistic alternatives have been proposed for the enzyme-catalyzed, electrophilic cyclization of farnesyl pyrophosphate to the tricyclic sesquiterpene alcohol patchoulol, which is the characteristic component of the essential oil of Pogostemon cablin (patchouli). These alternatives include schemes involving deprotonation-reprotonation steps and the intermediacy of the monocyclic and bicyclic olefins germacrene and bulnesene, respectively, and involving a 1,3-hydride shift with only tertiary cationic intermediates and without any deprotonation-reprotonation steps. Analytical studies, based on analyses of P. cablin leaf oil at different stages of plant development, and in vivo time-course investigations, using 14CO2 and [14C]sucrose, gave no indication that germacrene and bulnesene were intermediates in patchoulol biosynthesis. A soluble enzyme system from P. cablin leaves was prepared, which was capable of converting farnesyl pyrophosphate to patchoulol, and isotopic dilution experiments with both labeled and unlabeled olefins were carried out with this system to confirm that sesquiterpene olefins did not participate as fre intermediates in the transformation of the acyclic precursor to patchoulol. Patchoulol derived biosynthetically from [12,13-14C;1-3H]farnesyl pyrophosphate was chemically degraded to establish the overall construction pattern of the product. Similar studies with [12,13-14C;6-3H]farnesyl pyrophosphate as a precursor eliminated deprotonation steps to form bound olefinic intermediates in the biosynthesis of patchoulol, while providing supporting evidence for the hydride shift mechanism.

Cell-Free System↗
PubMed1989

Partial purification and properties of geranyl pyrophosphate synthase from Lithospermum erythrorhizon cell cultures.

A prenyltransferase (EC 2.5.1.1) was isolated from cell cultures of Lithospermum erythrorhizon. The enzyme was purified 92-fold by subsequent chromatography on DEAE-Sephacel, phenyl-Sepharose, and Sephadex G-150. Geranyl pyrophosphate (GPP) was the sole product of the enzymatic reaction with dimethylallyl pyrophosphate and isopentenyl pyrophosphate as the substrates. The enzyme showed a molecular weight of 73,000, estimated by gel chromatography on Sephadex G-150, and an isoelectric point at pH 4.95, determined by analytical isoelectric focusing. It had an absolute requirement for a divalent cation with Mg2+ and Mn2+ being most effective. The enzyme was soluble rather than membrane-bound. The physiological role of this prenyltransferase probably is to supply GPP for the biosynthesis of shikonin. It is the first chain-length specific geranyl pyrophosphate synthase reported from eukaryotic cells.

Chemical Phenomena↗
PubMed1985

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis.

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.

Animals↗
PubMed1984

Pyrophosphate functions as phosphoryl donor with UDP-glucose-treated mammalian phosphofructokinase.

Phosphofructokinase of rabbit muscle, which is specific for nucleoside triphosphates such as ATP, dissociated and gained the capability to utilize pyrophosphate as phosphoryl donor following incubation with UDP-glucose. The pyrophosphate- and ATP-linked activities of UDP-glucose-treated muscle phosphofructokinase were promoted by a protein species that showed a molecular weight of 80 kDa (vs. 320 kDa for the untreated enzyme). In the presence of citrate, a known inhibitor of PFK, the pyrophosphate-dependent activity elicited by UDP-glucose treatment was activated by fructose-2,6-bisphosphate. On removal of the UDP-glucose by either dialysis or dilution, the treated enzyme reassociated and became ATP-specific. ATP, dithiothreitol, and fructose-2,6-bisphosphate stimulated reassociation. The results suggest that metabolite-mediated catalyst conversion, yielding an enzyme form capable of utilizing both ATP and pyrophosphate, takes place with the phosphofructokinases of animal tissues.

Adenosine Triphosphate↗
PubMed1995

Different analogues of farnesyl pyrophosphate inhibit squalene synthase and protein:farnesyltransferase to different extents.

The inhibitory potency of farnesyl pyrophosphate analogues was investigated on two farnesyl pyrophosphate-consuming enzymes: squalene synthase, a secondary regulation site in the cholesterol synthesis pathway, and protein:farnesyl transferase, which plays a role in the function of Ras-proteins. For the transferase determination a rapid in vitro assay, using Sepharose-bound Ras-peptides, was developed. The distinct farnesyl pyrophosphate analogues showed a different order of potency in the inhibition of these two enzymes. Using the farnesyl transferase assay with pre-p21Ha-ras as substrate the same result was obtained. The difference observed in the in vitro assays was also reflected in the inhibition of cholesterol synthesis, protein prenylation in general and Ha-ras farnesylation in Rat-1.H-ras13 cells, a rat fibroblast cell line that overproduces human p21Ha-ras. This work shows that farnesyl pyrophosphate analogues can be developed for specific inhibition of different processes such as cholesterol synthesis and protein prenylation.

Alkyl and Aryl Transferases↗
PubMed1978

The estimation of pyrophosphate in urine with uridine-5'-diphosphoglucose pyrophosphorylase.

The concentration of pyrophosphate in urine has been determined using the enzyme uridine-5'-diphosphoglucose pyrophosphorylase. The technique is relatively quick and easy to perform. Reproducibility of results was good, and results from recovery experiments were excellent. The mean concentration of pyrophosphate found in early morning urine samples from 14 normal adults was identical with that reported by other workers using an ion-exchange method to measure pyrophosphate. The pyrophosphate concentration was related approximately to the phosphate concentration.

Adult↗
PubMed1989

Energy transduction at the catalytic site of enzymes: hydrolysis of phosphoester bonds and synthesis of pyrophosphate by alkaline phosphatase.

Alkaline phosphatase from mouse intestinal epithelial cells catalyzes the synthesis of pyrophosphate from Pi during hydrolysis of either glucose 6-phosphate, ATP, ADP, inorganic pyrophosphate or p-nitrophenylphosphate. The rate of pyrophosphate synthesis is increased by MgCl2 and by decreasing the pH of the medium from 8.5 to 6.0. The data presented indicate that at the catalytic site of alkaline phosphatase the energies of hydrolysis of the phosphoserine residue and of pyrophosphate are different from those measured in aqueous solutions.

Adenosine Diphosphate↗
PubMed1992

Purification and properties of a pyrophosphate-dependent phosphofructokinase from Toxoplasma gondii.

Inorganic pyrophosphate-dependent phosphofructokinase was identified in toxoplasma gondii and purified to near homogeneity from the crude extracts. The purified enzyme displayed one major protein band which coincided with enzyme activity on nondenaturing polyacrylamide gel electrophoresis. This phosphofructokinase had a molecular weight of 100,000 determined by gel filtration and was composed of one type of subunit with the molecular weight of 45,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a homodimer. Some kinetic parameters of the purified enzyme were investigated in the forward and the reverse directions. The substrate saturation curves for fructose 6-phosphate and pyrophosphate were all hyperbolic. The apparent Km values for fructose 6-phosphate and for pyrophosphate were 2.7 x 10(-4) M and 3.3 x 10(-5) M respectively. Kinetics for Fru-1,2-P2 and for Pi in the reverse reaction were also hyperbolic. The activity of this enzyme was magnesium-dependent. Nucleoside triphosphates and polyphosphates did not serve as phosphate donor and the enzyme activity was not altered in the presence of any of these nucleotides. As in the case of pyrophosphate-dependent phosphofructokinases from other anaerobic eukaryotes the Toxoplasma enzyme was not activated by fructose 2,6-biphosphate.

Animals↗
PubMed1975

Effect of adenine nucleotides and pyrophosphate on the exchange of transferrin-bound carbonate.

1. ATP, ADP and pyrophosphate accelerate the exchange of carbonate of the transferrin-iron-carbonate ternary complex, while AMP, cyclic AMP and phosphate have no effect. 2. ATP promotes carbonate exchange without removing iron from transferrin, whereas pyrophosphate effectiely attacks both the anion and iron components of the ternary complex. 3. Transferrin readily takes over iron from its ATP or pyrophosphate complex. 4. Neither ATP nor pyrophosphate can substitute for carbonate of the ternary complex. These results fit in well with the concept that ATP may play a direct role in the iron uptake by reticulocytes.

Adenine Nucleotides↗
PubMed1996

In vivo positive effects of exogenous pyrophosphate on Escherichia coli cell growth and stationary phase survival.

We have studied the effect of exogenous pyrophosphate on growing cells of Escherichia coli. In the presence of 10 mM of pyrophosphate, the entry into the stationary phase was delayed and thus a significant increase in the growth yield was observed (25 to 35%) when the bacteria were grown in glucose minimal medium. Furthermore, the synthesis of 52 polypeptides was affected, as demonstrated by two-dimensional electrophoresis. Among the 22 proteins identified by comparison with the E. coli gene-protein index and/or by microsequencing procedures, 15 were involved either in catabolic or anabolic pathways of the intermediary metabolism or in stress responses. Subsequent physiological experiments enabled us to conclude that pyrophosphate exerted a direct or indirect effect on bacterial growth by (1) conferring upon cells a better capacity to use carbon sources and (2) inducing biosynthetic processes. Finally, we show that exogeneous pyrophosphate enhanced the stationary phase survival of E. coli cells.

Autoradiography↗
PubMed1991

Dye-ligand and immobilized metal ion interaction chromatography for the purification of enzymes of prenyl pyrophosphate metabolism.

Dye-ligand and immobilized metal ion interaction chromatography were shown to be efficient techniques for the rapid batchwise fractionation, from crude plant extracts, of a series of enzymes of prenyl pyrophosphate metabolism. Isopentenyl pyrophosphate isomerase, two prenyltransferases, and a number of terpene cyclases (synthases) were readily adsorbed to Matrex Gel Red A (a dimeric triazine dye coupled to cross-linked agarose beads), and desorbed in good yield with relatively high concentrations of KCl and increasing pH. Although all of these enzymes exhibit the common feature of employing a pyrophosphorylated substrate, selective elution could not be achieved with substrate or substrate analogues bearing a pyrophosphate function. Nor could the strong binding of these enzymes to triazine dyes be attributed solely to metal ion interactions or to hydrophobic effects. In a similar way, the isomerase, the prenyltransferases, and all of the terpene cyclases bound to a column of iminodiacetate-immobilized Ni(II) and were desorbed in relatively high fold purity with 15 mM imidazole. Although all of these enzymes bear accessible histidine residues, the interactions with the chelated metal ion were not sufficiently different to permit selective enzyme desorbtion by imidazole gradient elution. However, the use of columns charged with Zn(II) or Co(II) did allow some separation of the different cyclase and transferase types. While empirical in nature, these techniques offer simple, effective, and high-capacity methods for the preliminary concentration and purification of a group of enzymes that utilize prenyl pyrophosphate intermediates of isoprenoid biosynthesis.

Chromatography, Affinity↗
PubMed1995

Direct presentation of nonpeptide prenyl pyrophosphate antigens to human gamma delta T cells.

Human V gamma 2V delta 2+ T cells recognize mycobacterial nonpeptide antigens, such as isopentenyl pyrophosphate, and their synthetic analogs, such as monoethyl phosphate, through a TCR-dependent process. Here, we examine the presentation of these antigens. V gamma 2V delta 2+ T cells recognized secreted prenyl pyrophosphate antigens in the absence of other accessory cells but, under such conditions, required T cell-T cell contact. Recognition required neither the expression of classical MHC class I, MHC class II, or CD1a, CD1b, and CD1c molecules, nor MHC class I or class II peptide loading pathways. Fixed accessory cells also presented the prenyl pyrophosphate antigens to gamma delta T cells. Thus, in contrast with the presentation of conventional peptide antigens, protein antigens, and superantigens to alpha beta T cells, prenyl pyrophosphate antigens are presented to gamma delta T cells through a novel extracellular pathway that does not require antigen uptake, antigen processing, or MHC class I or class II expression. This pathway allows for the rapid recognition of bacteria by gamma delta T cells and suggests that gamma delta T cells play a role in the early response to bacterial infection.

Antigen Presentation↗
PubMed2006

Characterization of the farnesyl pyrophosphate synthase of Trypanosoma cruzi by homology modeling and molecular dynamics.

Chagas' disease, caused by the Trypanosoma cruzi parasite, is one of the largest public health problems in the Western hemisphere, with 16-18 million people infected, and approximately 100 million people at risk. Many efforts towards the development of targeted antiparasitic agents have recently been described. Of interest, bisphosphonates, pyrophosphate analogs in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the growth of T. cruzi. The enzyme T. cruzi farnesyl pyrophosphate synthase (TcFPPS) involved in the mevalonate pathway, has been recently identified as the target of bisphosphonates. The protein has 362 amino acids and a molecular mass of 41.2 kDa. Several sequence motifs found in other FPPSs are present in TcFPPS. In this study we have modeled the structure of TcFPPS based on the structure of the avian FPPS. We have characterized the interaction with its substrates, isopentyl pyrophosphate and dimethylallyl pyrophosphate, and the mechanism of inhibition by the potent bisphosphonate risedronate (K(i) of 0.032+/-0.002 microM) by means of molecular dynamics techniques. We propose that homorisedronate, which has an extra methylene and a K(i) of 8.17+/-1.36 microM, does not form strong hydrogen bonds with TYR 211 and THR 208, which may be responsible for its lower activity as compared to risedronate. Moreover, we were able to reproduce the structural changes that occur upon the binding of the third Mg2+ to the active site of the protein. Taken together, our results provide a structural model for the design of novel inhibitors that may prove useful for the treatment of Chagas' disease.

Amino Acid Sequence↗
PubMed1977

Mechanisms contributing to myocardial accumulation of technetium-99m stannous pyrophosphate after coronary arterial occlusion.

The relation between the accumulation of pyrophosphate and technetium-99m in myocardium with reversible and irreversible ischmic injury was studied in dogs subjected to transitory or persistent coronary arterial occlusion. Among four dogs with coronary occlusion maintained for less than 20 minutes, none had either increased MB creatine kinase (CK) (the "myocardial" CK isoenzyme) activity serum or a positive 99mTc stannous pyrophosphate image. Seven dogs with coronary occlusion maintained for 30 or more minutes had elevated serum MB CK activity, and five of the seven had positive (abnormal) images. Thus, although false negative images may occur occasionally despite myocardial damage, both increased serum MB CK and abnormal images generally accompanied prolonged coronary occlusion. In contrast, ischemia without infarction was not associated with abnormal images. Both 99mTc and 32P labeled pyrophosphate were accumulated extensively and proportionally in myocardium from zones of infarction, and uptake of both tracers was comparable although modest in isolated mitochondria. Similar results were obtained after myocardial infarction in animals with induced profound leukopenia. Thus, phagocytosis of the radiopharmaceutical agent by leukocytes migrating into the infarct is not an essential mechanism accounting for uptake. These results indicate that abnormal images reflect uptake of pyrophosphate, associated with 99mTc, by irreversibly injured myocardium rather than leukocytic infiltration involved in the inflammatory response in the heart.

Animals↗
PubMed2002

Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s(-1) but product release limits the steady-state rate at 0.02 s(-1) [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 degrees C was 0.005 s(-1) under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C40 and the rate of its conversion to C45 was negligible. Under single-turnover condition with 10 microM OPPs-FPP complex and 1 microM IPP, only the C20 was formed rather than C20-C40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 degrees C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.

Alkyl and Aryl Transferases↗
PubMed2000

Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.

Alkyl and Aryl Transferases↗
PubMed1994

Mechanism of mevalonate pyrophosphate decarboxylase: evidence for a carbocationic transition state.

Mevalonate pyrophosphate decarboxylase catalyzes the decarboxylation of mevalonate pyrophosphate to isopentyl pyrophosphate. The mechanism of action of this enzyme was investigated to elucidate the mechanism of inhibition by 3-hydroxy-3-(fluoromethyl)-5-pyrophosphopentanoic acid (II). It was previously found that II is a competitive inhibitor (Ki = 0.01 microM) of the enzyme reaction [Reardon, J.E., & Abeles, R.H. (1987) Biochemistry 26, 4717-4722; Nave, J.F., d'Orchymont, H., Ducep, J.B., Piriou, F., & Jung, M.J. (1985) Biochem. J. 227, 247-254]. We have now observed that II is decarboxylated 2500-fold more slowly than mevalonate pyrophosphate (3-hydroxy-3-methyl-5-pyrophosphopentanoic acid, I). The enzyme was exposed to saturating concentrations of II and ATP and then passed through a Penefsky column to remove excess substrate. The enzyme was denatured immediately upon emerging from the Penefsky column. Nearly 1 equiv of both 3-phospho-3-(fluoromethyl)-5-pyrophosphopentanoic acid and ADP was bound to the enzyme. 3-Hydroxy-5-pyrophosphopentanoic acid (III) is phosphorylated at the secondary hydroxyl group and released from the enzyme without decarboxylation. This reaction is 30-fold slower than the rate of decarboxylation of I. The introduction of the 3-fluoromethyl group as well as the removal of the 3-methyl group results in low rates of decarboxylation. These substrate analogs have decreased electron density relative to the tertiary carbon of the substrate. Therefore, the transition state of the decarboxylation step has considerable carbocationic character. Further support for the carbocationic transition state is provided by the finding that N-methyl-N-carboxymethyl-2-pyrophosphoethanolamine (IV) inhibits the enzyme reaction with Ki = 0.75 microM. IV is probably a transition-state analog in which the positively charged nitrogen atom is analogous to the carbocation.

Carboxy-Lyases↗
PubMed1981

Structural changes in synthetic myosin minifilaments and their dissociation by adenosine triphosphate and pyrophosphate.

Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.

Adenosine Triphosphate↗