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PubMed1978

Structural features of normal and complemented forms of the Neurospora isopropylmalate isomerase.

The isopropylmalate isomerase (EC 4.2.1.33) of Neurospora crassa is a globular protein consisting of a single polypeptide chain with a molecular weight of about 90,000. The isomerase cannot easily be freed of a contaminating protease which cleaves the enzyme into two major fragments, one of approximately 56,000 and the other 37,000 daltons. This suggests that the folded polypeptide chain may contain some hinge point or loop exposed on the surface which makes it susceptible to proteolytic attack. Most of the isomerase activity extracted from the wild-type strain is in monomer form. However, a small fraction of the activity in crude extracts is found in multimeric aggregates, and the active isomerase extracted from complementing leu-2 heterokaryons consists entirely of dimers and higher multimers. These observations suggest that, though active as a monomer, a significant fraction of the normal enzyme might be organized in multimeric form within the cell.

Amino Acids↗

PubMed2001

A novel assay for fungal ketol-isomerase activity.

2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glutamate from fructose-6-phosphate and glutamine and plays an important role in chitin synthesis in fungi. We have established a new assay for fungal ketol-isomerase activity that is amenable to high throughput screening to identify enzyme inhibitors. Aspergillus fumigatus crude lysate was incubated with substrates and after incubation, reactions were terminated. Glutamate dehydrogenase, nitro blue tetrazolium chloride, phenazine methosulfate and beta-NAD were added and the amount of glutamate formed by ketol-isomerase activity was determined by measuring OD585nm. A feedback inhibitor, UDP-N-acetylglucosamine, of fungal ketol-isomerase was successfully detected by this assay (IC50=0.48 mM). In a pilot scale screening, an active extract from an extremophilic bacterium was found, and the extract showed antifungal activity against A. fumigatus, Candida albicans and C. glabrata.

Antifungal Agents↗

PubMed1975

Purification of yeast alpha-isopropylmalate isomerase. High ionic strength hydrophobic chromatography.

alpha-Isopropylmalate isomerase, the second enzyme specific for leucine biosynthesis, can be purified from extracts of yeast utilizing a chromatographic procedure that allows separation of proteins in the presence of high concentrations of (NH4)2SO4. The purification procedure utilizes the stabilizing effect of glycerol and (NH4)2SO4 on the isomerase and their opposing effects on protein retention on valine-Sepharose and leucine-Sepharose. The method effectively separates the isomerase from fumarase, a stable internal marker protein that was co-purified in early steps. High ionic strength hydrophobic chromatography, based on differential retention as a function of the length of the hydrophobic sidearm and ionic strength, yields approximately 200-fold purified alpha-isopropylmalate isomerase and may be of general utility in purifying unstable enzymes requiring high ionic strength.

Chromatography, Affinity↗

PubMed1993

Halothane hepatitis patients have serum antibodies that react with protein disulfide isomerase.

Clinical and laboratory evidence suggests that the fulminant liver failure sometimes associated with the inhalation anesthetic halothane may be an immune-mediated toxicity. Most importantly, the vast majority of patients with a clinical diagnosis of halothane hepatitis have serum antibodies, which react with one or more specific liver microsomal proteins that have been covalently altered by the trifluoroacetyl chloride metabolite of halothane. The serum antibodies are specific to halothane hepatitis patients and are not seen in sera of patients with other types of liver pathology. In this study, a 57-kD trifluoroacetylated liver microsomal neoantigen associated with halothane hepatitis and native 57-kD protein were purified from liver microsomes of halothane-treated and -untreated rats, respectively. When the purified trifluoroacetylated 57-kD and native 57-kD proteins were used as test antigens in an enzyme-linked immunosorbent assay, serum antibodies from halothane hepatitis patients (n = 40) reacted with both of these proteins to a significantly greater extent than did serum antibodies from control patients (n = 32). On the basis of its apparent monomeric molecular mass, isoelectric point and NH2-terminal amino acid and tryptic peptide sequences, the 57-kD protein has been identified as rat liver protein disulfide isomerase. Antibodies raised against rat liver protein disulfide isomerase also reacted with a protein of approximately 58-kD in human liver microsomes. The results of this investigation suggest that trifluoroacetylated protein disulfide isomerase is one of the immunogens associated with halothane hepatitis. In certain patients it might lead either to specific antibodies or, possibly, to specific T cells, which could be responsible for halothane hepatitis.

Antibodies↗

PubMed1993

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.

Disulfides↗

PubMed2003

Gene regulation in response to protein disulphide isomerase deficiency.

We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue, revealed a cis-acting element responding to deficiency of protein disulphide isomerase activity (designated CERP). The presence of the sequence element is necessary and sufficient for the upregulation in response to disulphide isomerase deficiency, as measured by a minimal promoter containing the CERP element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes.

Base Sequence↗

PubMed1995

Peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase and thus possess all enzymes required for the beta-oxidation of unsaturated fatty acids by a novel reductase-dependent pathway.

The presence of delta 3,5,delta 2,4-dienoyl-CoA isomerase in peroxisomes was demonstrated by determining the subcellular distribution of this enzyme in rat liver. The peroxisomal and mitochondrial forms of the isomerase exhibit similar chain length specificities and they are homologous as indicated by the recognition of the peroxisomal 66-kDa enzyme by an antiserum raised against the mitochondrial 32-kDa isomerase. This report demonstrates that peroxisomes contain all enzymes required for the beta oxidation of unsaturated fatty acids with odd-numbered double bonds by a novel pathway in which double bonds are reductively removed by the NADPH-dependent 2,4-dienoyl-CoA reductase.

Animals↗

PubMed2001

Solution structure of the human parvulin-like peptidyl prolyl cis/trans isomerase, hPar14.

The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30 % sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36-131 of hPar14, which comprises a four-stranded beta-sheet and three alpha-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of alpha3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1.

Amino Acid Sequence↗

PubMed1990

Determination of aconitate isomerase in plants.

We have developed a method for measuring aconitate isomerase (EC 5.3.3.7) in plants which depends on the release of tritium from labeled trans-aconitate. The released tritium is separated from labeled aconitate by passage through a column of strong anion-exchange resin. This method is more sensitive, simpler, and more specific than previous methods, especially with crude extracts. The validity of the method was demonstrated by a comparison of the quantity of tritium released with the amount of cis-aconitate isomerized and this comparison demonstrated an isotope effect. Aconitase did not interfere. The method was tested by measuring aconitate isomerase in crude extracts of several higher plant species and tissues and these analyses showed that wheat and corn have more aconitate isomerase than the other species tested.

Aconitate Hydratase↗

PubMed1977

A reinvestigation of the mechanism of Pseudomonas testosteroni delta 5-3-ketosteroid isomerase.

The mechanism of the isomerisation of delta 5-3,17-androstenedione by the isomerase (3-oxosteroid delta 4-delta 5-isomerase, EC 5.3.3.1) of Pseudomonas testosteroni has been reinvestigated with delta 5-[4-beta-2H]androstenedione as substrate in H2O and delta 5-androstenedione in 2H2O. A precise localisation of the label in delta 4-androstenendione has revealed that the previously reported 4 beta leads to 6 beta deuterium transfer accounts for only a part of the reaction. Along with this process, removal of the 4 alpha proton is also occurring. This has already been observed with mammalian isomerases. Hence the assumed difference in mechanism between the bacterial and mammalian enzymes is very unlikely.

Androstenedione↗

PubMed1985

The refolding of urea-denatured ribonuclease A is catalyzed by peptidyl-prolyl cis-trans isomerase.

The refolding of urea-denatured ribonuclease A was measured at 0.31-3.1 mol . l-1 urea in the presence of various concentrations of peptidyl-prolyl cis-trans isomerase isolated from pig kidney. The rate of the slow CT-phase in the refolding reaction was found to be sensitive to this enzyme. A rate enhancement proportional to the isomerase activity has been observed. The activity of the enzyme was assayed with Glt-Ala-Ala-Pro-Phe-4-nitroanilide. The catalytic activity of the isomerase against unfolded ribonuclease is suppressed after preincubation of the enzyme with 0.001 mol . l-1 Cu2+, 0.01 mol . l-1 H+ and by heat inactivation. The results indicate the involvement of the cis/trans interconversion of proline peptide bonds during the refolding of ribonuclease A.

Amino Acid Isomerases↗

PubMed1994

Prolyl isomerase requirement for the expression of functional homo-oligomeric ligand-gated ion channels.

Ligand-gated ion channel subunits show a striking abundance of highly conserved proline residues. We, therefore, tested the hypothesis that peptidyl-prolyl isomerases may be involved in the maturation of these channels. Cyclosporin A, a selective blocker of a ubiquitous isomerase cyclophilin, reduced the surface expression in Xenopus oocytes of functional homo-oligomeric receptors containing nicotinic acetylcholine receptor subunit alpha 7 without blocking alpha 7 polypeptide synthesis. This effect could be generalized to the homo-oligomeric 5-hydroxytryptamine type 3 receptor but not to the hetero-oligomeric muscle nicotinic receptor. An alpha 7 receptor could be rescued from cyclosporin A blockade by coexpressed muscle non-alpha subunits. The effect of cyclosporin A was reversed by overexpression of exogenous rat brain cyclophilin. These findings indicate that cyclophilins may play a critical role in the maturation of homo-oligomeric receptors, acting directly or indirectly as prolyl isomerases or as molecular chaperones.

Amino Acid Isomerases↗

PubMed1996

Cloning, expression and genomic organization of human placental protein disulfide isomerase (previously identified as phospholipase C alpha).

Phosphoinositol-specific Phospholipase C plays an important role in transducing receptor generated signals to the rest of the cell. A cDNA encoding a phospholipase has been described (Bennett et al., 1988, Nature 334, 268-270). However it is probable that this cDNA in fact encodes a protein disulfide isomerase. Since the original work suggested that this enzyme was important in the reproductive tract we sort to clone, sequence, express and characterize the recombinant protein isolated from the placenta. We have cloned and sequenced the cDNA encoding the human homolog of this cDNA from human placenta, although the mRNA was widespread in the female reproductive tract. We have transiently expressed it in both COS cells and also 1BR fibroblasts. Cell lysates were assayed for increased phospholipase activity and protein disulfide activity. We describe the entire cDNA sequence which is highly conserved between species. We have also cloned a portion of the genomic gene and described the intron/exon boundaries. In vitro translation of this cDNA showed that it encoded a protein of 61 kD with a cleavable signal peptide. Transient expression showed the protein produced had no phospholipase activity but did show protein disulfide isomerase activity. The expression work shows that this cDNA indeed encodes a protein disulfide isomerase and not a phospholipase. The nucleotide sequence shows marked conservation of the coding and regulatory regions which may suggest that this enzyme has evolved to perform a highly specialized function.

Base Sequence↗

PubMed2006

Mutation of Lys242 allows Delta3-Delta2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity.

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Delta3-Delta2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k(cat) value of 1.0 s(-1) for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.

Amino Acid Sequence↗

PubMed2001

The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli.

To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co-expression. When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies. Co-expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Co-expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell. Co-expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl-prolyl cis/trans isomerase activity was 1% of wild-type, gave the same results as wild-type TF. This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.

Adenylate Kinase↗

PubMed1997

Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate.

The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.

Amino Acid Isomerases↗

PubMed1997

Mutant human protein disulfide isomerase assists protein folding in a chaperone-like fashion.

Human protein disulfide isomerase with an extra 10 amino acid residues of AEITRIDPAM at the N-terminal was expressed in E. coli as a soluble protein comprising 20% of total cell proteins, and was purified to near homogeneity through one step of DEAE-Sephacel chromatography. The mutant enzyme, which had the same CD spectrum and comparable disulfide isomerase and thiol-protein oxidoreductase activities with that of the wild type human and bovine protein disulfide isomerases, also showed chaperone-like activity in stimulating the refolding of proteins containing no disulfide bond. The overall yield of the active product is about 20 mg 1-1 culture.

Amino Acid Sequence↗

PubMed1992

Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate.

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.

Amino Acid Sequence↗