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PubMed2002

Functional characterization of Delta3,Delta2-enoyl-CoA isomerases from rat liver.

The degradation of unsaturated fatty acids by beta-oxidation involves Delta(3),Delta(2)-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of enoyl-CoAs and the 2,5 --> 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis --> 2-trans isomerization; ECI has a preference for the 3-trans --> 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 --> 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis --> 2-trans and 2,5 --> 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans --> 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis --> 2-trans and 3-trans --> 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 --> 3,5 isomerization.

Animals↗
PubMed1990

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity.

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.

Amino Acid Sequence↗
PubMed1988

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism.

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.

Binding Sites↗
PubMed1984

Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme.

Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.). Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0. This preparation was used to raise rabbit anti-(chalcone isomerase) serum. Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with [35S]methionine in elicitor-treated cultures of P. vulgaris. Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor. Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000. However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity. Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme. It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity. The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O [Dixon et al. (1983) Planta (Berl.) 159, 561-569].

Chromatography↗
PubMed1990

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.

Animals↗
PubMed1972

Multiple forms of glucosephosphate isomerase in maize.

Three apparently different glucosephosphate isomerases are found in the developing seeds of maize (Zea mays L.). Glucosephosphate isomerase I is found in both the endosperm and embryo. It is separable by column chromatography from glucosephosphate isomerase II of the developing endosperm and glucosephosphate isomerase III of the developing embryo and is further distinguished from them by heat stability, temperature activation, and relative insensitivity to the presence of zinc ions in the reaction mixture. Glucosephosphate isomerases II and III elute in the same fractions from diethylaminoethyl cellulose columns but are distinguished by electrophoretic mobility and reaction to the presence of adenosine 5'-triphosphate in the reaction mixture. All three isomerases give multiple banding patterns on electrophoresis. An extensive investigation of the conditions generating additional electrophoretic species and chromatographically separable minor activity peak (Ia) from glucosephosphate isomerase I has shown that these transformations are enhanced by dialysis, column chromatography, ammonium sulfate fractionation, and treatment with urea. The transformations are retarded by the presence of mercaptoethanol during these operations. We concluded that the multiple banding pattern seen on electrophoresis of glucosephosphate isomerase I prepared by certain procedures is artifactual. In germinating seeds of maize, glucosephosphate isomerases I and III are detectable, but II is not. It is possible that glucosephosphate isomerase II specifically catalyzes a step in starch biosynthesis.

Journal Article↗
PubMed1984

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 X 10(-3) M 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 Km of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 X 10(-6).

Carbon-Carbon Double Bond Isomerases↗
PubMed1990

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile.

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae [Anderson, M. S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175]. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea [Muehlbacher, M., & Poulter, C. D. (1988) Biochemistry 27, 7315-7328]. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.

Amino Acid Sequence↗
PubMed1996

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Blood Platelets↗
PubMed1989

Identification and mechanism of action of phospholipids capable of modulating rat testicular microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase activity in vitro.

Rat testicular 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) converts pregnenolone to progesterone. The enzyme is localized to the microsomal membranes of testicular homogenates, and treatment of the microsomes with phospholipases causes a reduction in 3 beta-HSD-Isomerase activity. The relationship between the membrane microenvironment and 3 beta-HSD-Isomerase activity was investigated by adding phospholipids of known structure to microsomal incubations and determining the effects on the conversion of pregnenolone to progesterone. Phosphatidylcholines with saturated acyl chains of 8, 10, 12, and 14 carbon atoms, or unsaturated chains, were extremely inhibitory to 3 beta-HSD-Isomerase, causing reductions in specific activity to 10-40% of the control value. Furthermore, the inhibition appeared to be caused primarily by a reduction in the active enzyme concentration (Vmaxapp). Phosphatidylcholines with longer saturated acyl chains were without effect. Phosphatidylserine and phosphatidic acid were activators of 3 beta-HSD-Isomerase. These phospholipids decreased the Kmapp value (to 21% and 43% of control values, respectively), suggesting that the enhancement of 3 beta-HSD-Isomerase activity was through an active-site-oriented effect. Furthermore, for phosphatidic acid to activate 3 beta-HSD-Isomerase, saturated acyl chains of 16 carbon atoms were necessary, other configurations being slightly inhibitory. The remarkable specificity for certain phospholipid configurations and the different effects of these membrane components suggest that androgen biosynthesis may be regulated by changes in the phospholipid microenvironment of 3 beta-HSD-Isomerase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Genetic control of corticosteroid side-chain isomerase activity in the mouse.

The corticosteroid side-chain isomerases of mammalian liver catalyze the interconversion of the ketol and aldol side chains. In the mouse, isomerase was low in C57BL/6 (B6) mice (130 pmol/mg protein . 2 h) and high in BALB/c (C) mice (230 pmol/mg protein . 2 h). From analysis of hybrids between B6 and C and of backcrosses of these hybrids to B6, it was concluded that isomerase levels are controlled by a single autosomal gene dominant for high activity. The distribution of high and low isomerase levels in a series of CXB/By recombinant inbred strains of mice was consistent with linkage of the isomerase gene to H-2. Congenic BALB.B mice (H-2b haplotype from C57BL/10) had low isomerase activities corresponding to C57BL/10, not the high activity of the background strain BALB/c(H-2d). Similarly, BN10.D2 congenic mice (H-2d haplotype from the DBA/2 strain) had high activity characteristic of DBA/2. In the (C X B6)F1, (C X BALB.B)F1 and (B10 X B10.D2)F1 hybrids, all of which are H-2d/H-2d heterozygotes, isomerase activity was high. The association of isomerase levels with H-2 type was further confirmed in mice of the following backcrosses: (C X BALB.B)F1 X BALB.B, (C X B6)F1 X B6 and (B10 X B10.D2)F1 X B10. H-2b/H-2b homozygous segregants had consistently low activity and H-2b/H-2d heterozygous segregants had consistently high activity. It was concluded that the level of corticosteroid side-chain isomerase activity in mouse liver is controlled by a gene(s) in the region of the H-2 locus on chromosome 17.

Animals↗
PubMed1987

Biochemical characterization of the retinoid isomerase system of the eye.

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.

Animals↗
PubMed2005

Assay of disulfide oxidase and isomerase based on the model of hirudin folding.

Oxidative folding of fully reduced hirudin (R-Hir, six cysteines) undergoes two distinct stages. A first stage of nonspecific disulfide formation promoted by oxidase converts R-Hir to form 3-disulfide scrambled hirudins (X-Hir) as obligatory intermediates. A second stage of disulfide shuffling catalyzed by isomerase converts X-Hir to the native hirudin (N-Hir). The model of hirudin folding is utilized here to develop an assay system for measuring the activity of disulfide oxidase and isomerase, using high-performance liquid chromatography (HPLC) quantification of R-Hir, X-Hir, and N-Hir. The oxidase assay measures the ability of an oxidase to promote R-HirX-Hir conversion. The molar specific activity is expressed as mol ofR-Hir decrease per mol of oxidase per min. The isomerase assay measures the ability of an isomerase to catalyze X-HirN-Hir transformation. The molar specific activity is expressed as mol ofN-Hir increase per mol of isomerase per min. Alternatively, the recovery of N-Hir in the isomerase assay can be determined by its alpha-thrombin inhibitory activity. Using both HPLC and activity-based assay, we have measured the relative oxidase and isomerase activity of reduced and oxidized glutathione, Cys, Cys-Cys, and reduced and oxidized protein disulfide isomerase (PDI). The molar specific activity of reduced PDI was shown to be 0.1+/-0.01 U, which is consistent with documented data obtained by the scrambled RNase-A-based assay. These proposed assay methods provide alternatives to the limited option of methodologies currently available for measuring oxidase and isomerase activities. A major merit of the proposed assay system is the potential to accommodate the analysis of biological samples.

Chromatography, High Pressure Liquid↗
PubMed1995

An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 microM, Vmax = 2.1 mumol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.

Adenosine Diphosphate↗
PubMed2004

The contributions of protein disulfide isomerase and its homologues to oxidative protein folding in the yeast endoplasmic reticulum.

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 +/- 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.

Endoplasmic Reticulum↗
PubMed1991

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody.

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.

Animals↗
PubMed1989

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator.

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.

Animals↗
PubMed2000

Insect melanogenesis. III. Metabolon formation in the melanogenic pathway-regulation of phenoloxidase activityy by endogenous dopachrome isomerase (decarboxylating) from Manduca sexta.

Tyrosinase initiates melanogenesis in a variety of organisms. The nature of melanin formed is modified subsequently by dopachrome isomerase and other melanogenic proteins. Earlier, we reported the partial purification of dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta and demonstrated the generation of a new quinone methide intermediate during melanogenesis (Sugumaran, M., and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). In this paper, we report the purification of this enzyme to homogeneity and a novel inhibition mechanism for regulation of phenoloxidase activity. The activity of phenoloxidase isolated from M. sexta was markedly inhibited by purified dopachrome isomerase. In turn, phenoloxidase also reciprocated by inhibiting the isomerase activity. Preformed dopaminechrome did not serve as the substrate for the isomerase; but dopaminechrome that generated in situ by phenoloxidase was readily converted to melanin pigment by the phenoloxidase/isomerase mixture. Furthermore, the isomerase, which has a molecular weight of about 40,000 in native state, exhibited retardation during affinity electrophoresis on sodium dodeyl sulfate (SDS)-polyacrylamide gel electrophoresis gel copolymerized with tyrosinase and migrated with a molecular weight of 50,000, indicating complex formation with phenoloxidase. Electrophoresis of pupal cuticular extract on polyacrylamide gel, followed by activity staining revealed the presence of a protein band carrying both phenoloxidase and isomerase activity. Accordingly, a high-molecular-weight melanogenic complex was isolated from the pharate cuticle of M. sexta. The complex catalyzed the generation of melanochrome from dopa, while the free phenoloxidase produced only dopachrome from the same substrate. When the complex was treated with trace amounts of SDS, which inhibited the activity of dopachrome isomerase present in the complex, then only the conversion of dopa to dopachrome was observed. These studies confirm the formation of a melanogenic complex between phenoloxidase and dopachrome isomerase. By forming a complex and regulating each other's activity, these two enzymes seem to control the levels of endogenous quinones.

Animals↗