Isopentenyl pyrophosphate isomerase from liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6.0 and is activated by Mn(2+) in preference to Mg(2+). The K(m) value for isopentenyl pyrophosphate is 4x10(-6)m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

The purification of 3,3-dimethylallyl- and geranyl-transferase and of isopentenyl pyrophosphate isomerase from pig liver

The enzyme catalysing the synthesis of farnesyl pyrophosphate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate, or from geranyl pyrophosphate and isopentenyl pyrophosphate, has been purified 100-fold from homogenates of pig liver. The enzyme has optimum pH 7.9 and requires Mg(2+) as activator in preference to Mn(2+); it is inhibited by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate and phosphate ions in addition to the products of the reaction, inorganic pyrophosphate and farnesyl pyrophosphate. From product-inhibition studies of the geranyltransferase reaction, the order of addition of substrates to and release of products from the enzyme has been deduced: geranyl pyrophosphate combines with the enzyme first, followed by isopentenyl pyrophosphate. Farnesyl pyrophosphate dissociates from the enzyme before inorganic pyrophosphate. The existence of isopentenyl pyrophosphate isomerase in liver is confirmed. Methods for the preparation of the pyrophosphate esters of isopentenol, 3,3-dimethylallyl alcohol, geraniol and farnesol are also described.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Isoprenoid enzyme systems of silkworm. I. Partial purification of isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and geranylgeranyl pyrophosphate synthetase

Isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and geranylgeranyl pyrophosphate synthetase were detected in cell-free extracts of Bombyx mori and were partially purified by hydroxyapatite and Sephadex G-100 chromatography. Two forms of farnesyl pyrophosphate synthetase were chromatographically separated. They were designated as farnesyl pyrophosphate synthetases I and II in the order of their elution from hydroxyapatite. Both enzymes catalyzed the exclusive formation of (E,E)-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. However, they were not interconvertible, unlike the enzyme from pig liver. These two enzymes resembled each other in pH optima and molecular weights but differed in susceptibility to metal ions. Farnesyl pyrophosphate synthetase II was stimulated by Triton X-100 while synthetase I was inhibited by the same reagent.     

Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.     

Multiple forms of isopentenyl pyrophosphate isomerase of avian liver

Four kinds of isopentenyl pyrophosphate isomerase were separated from avian liver homogenates by DE-52 cellulose column chromatography. These enzymes were similar in terms of optimum pH (pH 6.6) and metal ion requirement (Mn2+). They all catalyzed the specific elimination of the pro-R hydrogen at C-2 of isopentenyl pyrophosphate. However, they showed different susceptibilities to iodoacetamide and beta-mercaptoethanol.     

Undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum: a dimeric protein

a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by preparative gel electrophoresis and Blue-agarose column chromatography. The Km's of the enzyme for its substrates t,t-farnesyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.13 and 1.92 microM, respectively. The molecular weight of the enzyme was estimated by molecular sieve chromatography and gradient centrifugation to be 56,000 +/- 4000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the protein was composed of a dimer of 30,000-Da subunits. The enzyme was inactivated by the arginine-specific reagents phenylglyoxal, butanedione and, cyclohexanedione, but this inactivation was not prevented by either of the substrates.     

Disequilibrium in the triose phosphate isomerase system in rat liver

1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.     

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.     

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.     

Synthesis of 10,11-dihydrofarnesyl pyrophosphate from 6,7-dihydrogeranyl pyrophosphate by prenyltransferase

The syntheses of 6,7-dihydrogeraniol and of its pyrophosphate are described. It is shown that this analogue of geranyl pyrophosphate is a substrate for liver prenyltransferase and that the product synthesized by this enzyme from it and isopentenyl pyrophosphate is 10,11-dihydrofarnesyl pyrophosphate. The K(m) value for 6,7-dihydrogeranyl pyrophosphate was determined to be 1.11+/-0.19mum as compared with 4.34+/-1.71mum for geranyl pyrophosphate. The maximum reaction velocity with the artifical substrate was, however, only about one-fourth of that observed with geranyl pyrophosphate. The binding of isopentenyl pyrophosphate to the enzyme was not affected by the artificial substrate.     

Immunoaffinity purification of rat liver transketolase: evidence for multiple forms of the enzyme

Rat liver transketolase (TK) has been purified, in a single step, by immunoaffinity chromatography on specific TK antibodies covalently linked to Sepharose 4B. The procedure described also involves the raising and isolation of rabbit TK antibodies to the conventionally purified enzyme [F. Paoletti (1983) Arch. Biochem. Biophys. 222, 489-496]. Affinity chromatography allows a 100-fold purification of TK from the cell cytosol and a recovery of about 70% of the original activity. The TK isolated has a specific activity of 2.7-3.2 at 25 degrees C and migrates as a single band on polyacrylamide gel electrophoresis at pH 9.1. Multiple forms of the enzyme, with distinct pI values in the range 7-8, have been detected in purified preparations by means of analytical isoelectric focusing and staining for TK. No addition of either Mg2+ or thiamine pyrophosphate is required for the activity of the enzyme which, in the native form, exhibits a molecular weight of about 139,000. Two moles of thiamine pyrophosphate can be resolved for each mole of enzyme. Affinity TK preparations are virtually free of glyceraldehyde-3-phosphate dehydrogenase, pentose-phosphate epimerase, and isomerase, although slight contamination by phosphohexose isomerase may occur.     

Purification and characterization of dimethylallyl pyrophosphate: aspulvinone dimethylallyltransferase from Aspergillus terreus

Dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase, the prenylation enzyme for the biosynthesis of aspulvinone pigments, has been purified from mycelia of Aspergillus terreus. The transferase catalyzed the transfer of the dimethylallyl moiety from dimethylallyl pyrophosphate to either of the two aromatic rings of aspulvinone E to give the mono- and diprenylated derivatives which were identified with the metabolites aspulvinone I and aspulvinone H, respectively. Aspulvinone G, another fundamental metabolite of this series, also acted as substrate to afford the corresponding diprenylated derivative, which is assumed to be a precursor for aspulvinone C, D, and F. The molecular weight of the enzyme was estimated to be 240 000--270 000 by gel filtration. Since the subunit molecular weight determined by NaDodSO4-polyacrylamide disc gel electrophoresis was 45 000, the native enzyme appears to be a hexomeric protein composed of identical molecular weight subunits. The apparent Km values for aspulvinone E, aspulvinone G, and dimethylallyl pyrophosphate were 13.7, 7.7, and 40.0 micron, respectively. The enzyme shows the maximum activity at pH 7.0, and no metal ion is necessary for the activation. Sulfhydryl blocking agents or mercaptoethanol has no effect. Bromophenol blue binds specifically to the transferase and strongly inhibits the enzyme activity.     

Crystal structure of type 2 isopentenyl diphosphate isomerase from Thermus thermophilus in complex with inorganic pyrophosphate

The N-terminal region is stabilized in the crystal structure of Thermus thermophilus type 2 isopentenyl diphosphate isomerase in complex with inorganic pyrophosphate, providing new insights about the active site and the catalytic mechanism of the enzyme. The PP i moiety is located near the conserved residues, H10, R97, H152, Q157, E158, and W219, and the flavin cofactor. The putative active site of isopentenyl diphosphate isomerase 2 provides interactions for stabilizing a carbocationic intermediate similar to those that stabilize the intermediate in the well-established protonation-deprotonation mechanism of isopentenyl diphosphate isomerase 1.     

A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10

beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp. produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The Streptomyces sp. beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C. The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8). Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases.     

Biosynthesis of presqualene pyrophosphate by liver microsomes

Microsomes from rat liver have been shown to synthesize a squalene precursor from farnesyl pyrophosphate. This intermediate is identical with presqualene pyrophosphate, a 30-carbon cyclopropane containing pyrophosphate ester that had previously been isolated from yeast. The squalene precursor was found to be tightly, but not covalently, bound to microsomes.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

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.