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PubMed1993

Limited proteolysis of triose-phosphate isomerase and characterization of the catalytically active peptide complex.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

Amino Acid Sequence↗
PubMed1977

Prostaglandin endoperoxide E isomerase from bovine vesicular gland microsomes, a glutathione-requiring enzyme.

Prostaglandin endoperoxide E isomerase catalyzes the isomerization of the endoperoxy group of prostaglandin H and produces prostaglandin E. The enzyme was solubilized with Tween 20 from bovine vesicular gland microsomes and purified 26-fold by successive column chromatography on DEAE-cellulose and omega-aminobutyl Sepharose 4B. The previously known requirement for glutathione was further investigated. The activity of freshly prepared enzyme was destroyed rapidly with a half-life of about 30 min at 24 degrees, pH 8.0. Various thiol compounds including glutathione protected the enzyme from such an inactivation. However, several findings indicated that glutathione was possibly specifically involved as a coenzyme in the isomerase reaction, but was not oxidized in a stoichiometric quantity. The enzyme also isomerized prostaglandin G at a rate approximately half of that of prostaglandin H.

Animals↗
PubMed1996

[The effect of intravenous glucose load on levels of glucose and fructose and activity of glucose-6-phosphate isomerase in serum of patients with subarachnoid hemorrhage].

The purpose of the study was to evaluate the effect of glucose load on changes of glucose and fructose concentrations in the blood and phosphohexose isomerase activity (PHI) in the serum of patients with subarachnoid haemorrhage. The investigation was applied to 20 patients with subarachnoid haemorrhage. The control group included 20 individuals. In patients with subarachnoid haemorrhage the intravenous load of hypertonic glucose solution resulted in the extended blood hyperglycaemia and the decrease of fructose concentration in final moments of test in the III, VII and XIV day of illness. The activity of phosphohexose isomerase remained normal both before and after the glucose load.

Blood Glucose↗
PubMed1998

Lowering the pH optimum of D-xylose isomerase: the effect of mutations of the negatively charged residues.

Streptomyces rubiginosus D-xylose isomerase catalyzes the reversible isomerization of D-glucose to D-fructose. The isomerization reaction is maximized in the alkaline region of pH 8.5-8.8. The amino acid residues around two active site histidines (His-54 and His-220) and on the surface of the enzyme were mutated to improve the catalytic efficiency at neutral pH. The mutations have been made by removing the negatively charged residues based upon the sequence comparison of other D-xylose isomerases and the hypothesis proposed by Russell and Fersht (1987). The effects of these substitutions on kinetic parameter, pH dependence, and thermostability were characterized. The kcat values for D56N and E221A mutants on D-glucose are increased by 30-40% over that of the wild-type enzyme at pH 7.3 and the increased activities are maintained between pH 6 and 7.5. However, the surface mutants D65A, D81A, and D163N/E167Q only show 40-60% of the wild-type activity over the entire pH range. The pH activity profiles of the mutants are broader than that of the wild-type enzyme. The optimum pHs and the pKa values for all the mutants are lowered by 0.5-0.8 and 0.1-0.5 units, respectively. The small delta(deltaG) and high Tm values for all the mutants indicate that there is no significant change in the hydrogen bond network in the active site by mutations. These results indicate that D56N and E221A are possible candidates as good catalysts for High-Fructose Corn Syrup (HFCS) production.

Aldose-Ketose Isomerases↗
PubMed1979

Proteolytic denaturation and methods of improving the stability of glucose isomerase preparations.

Evidence is provided in support of proteolytic denaturation of free and immobilized preparations of glucose isomerase from a Bacillus species. A number of methods to improve the stability with respect to proteolysis have been tested and their advantages as well as shortcomings are discussed. These methods include hollow-fiber treatment, gel permeation, thermal treatment, and addition of protease inhibitors. The half-life of the free and the cellulose acetate fiber-entrapped preparations of glucose isomerase can be significantly improved. For example, the hollow-fiber treatment can improve the half-life by an order of magnitude.

Acetates↗
PubMed1987

Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection.

The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A lambdagt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 10 recombinants with an antiserum to purified bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. Addition of elicitor to cell cultures resulted in the rapid accumulation of CHI mRNA, with maximum levels achieved 3-4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals were strikingly similar to those of mRNAs encoding two other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families encoding these two enzymes, chalcone isomerase is encoded by a single gene which is regulated by several environmental stimuli.

Journal Article↗
PubMed1975

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.

Alleles↗
PubMed1985

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment.

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Ethanol directly inhibits delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in rat testis interstitial cells.

Acute ethanol exposure has been demonstrated to inhibit testosterone synthesis both in vivo and in vitro; however, the precise step(s) affected is controversial. Using intact collagenase-dispersed interstitial cells or 10,000 xg supernatants of interstitial cell homogenates, studies were undertaken to determine whether ethanol specifically inhibited delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity. In both cellular preparations, varying concentrations of ethanol (2.2 - 652 mM) inhibited this enzyme activity. Because alcohol dehydrogenase activity was identified specifically in Leydig cells and because the inhibition of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity by concentrations of ethanol normally observed in circulation of alcoholic men (2.2 - 65 mM) could be reversed by saturating concentrations of NAD+ (0.2 mM) or by 4-methylpyrazole (2 mM), these results suggest that the mechanism of this inhibition is by limitation of available cofactor.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

The cDNA cloning and transient expression of a chicken gene encoding a 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase unique to major steroidogenic tissues.

Two cDNA clones, on containing a 2.7-kb and the other a 1.6-kb insert, were obtained from a chicken adrenal gland cDNA library by screening with a partial chicken cDNA fragment generated by PCR. Primers were used that corresponded to conserved regions among several published cDNA sequences of mammalian 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase (3 beta-HSD). Both clones contained the same open reading frame (ORF) encoding 377 amino acids (aa). The difference in their sizes was due to the use of different polyadenylation sites. The deduced aa sequence showed 54-57% overall identity with those of the human, macaque, bovine, mouse, rat and rainbow trout 3 beta-HSD. A 9-aa stretch (FYYISDDTP) is conserved among the chicken, mammalian and rainbow trout 3 beta-HSD, suggesting that it is essential for enzymatic activity. Northern blot hybridization demonstrated the presence of two types of mRNA, corresponding to the above two cDNA classes, in the adrenal gland, ovary and testis. Transient expression of the chicken ORF in COS-7 cells demonstrated coupled dehydrogenase and isomerase activities of 3 beta-HSD on three different substrates; pregnenolone, 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Among the Km values determined for the three substrates, that for dehydroepiandrosterone was the lowest, which was different from the case for human 3 beta-HSD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Evidence that biphasic effects of basic fibroblast growth factor on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans.

The present studies examined the effects of heparin, heparinase, insulin or insulin-like growth factor-I on basic fibroblast growth factor actions on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature rat Leydig cells. Treatment with basic fibroblast growth factor alone (0.025-20 ng/ml) for 2 days had a biphasic effect on enzyme activity, with lower concentrations (0.025-1 ng/ml) progressively inhibiting activity to approximately 20% of control, while higher concentrations (2.5-20 ng/ml) partially reversed the inhibitive effects. The inclusion of 10 micrograms/ml heparin, a concentration reported to inhibit growth factor binding to heparan sulfate proteoglycans, blocked the increase in enzyme activity elicited by higher growth factor concentrations, but had no effect on the progressive decline in activity due to lower concentrations. Concomitant treatment with heparinase I and III, which specifically hydrolyze heparan sulfate proteoglycans, had a similar effect. In addition, both insulin and insulin-like growth factor-I partially reversed the inhibition of enzyme activity due to treatment with 1 ng/ml basic fibroblast growth factor. These studies suggest that some basic fibroblast growth factor actions on cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans, and that both insulin and insulin-like growth factor-I can reverse the inhibitive effects on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed2004

Identification of the protein disulfide isomerase family member PDIp in experimental Parkinson's disease and Lewy body pathology.

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.

1-Methyl-4-phenylpyridinium↗
PubMed2005

Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action.

Immunophilin is the collective name given to the cyclophilin and FK506-binding protein families. As the name suggests, these include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FK506-binding proteins for the macrolactones FK506 and rapamycin. Both families, although dissimilar in sequence, possess peptidyl-prolyl cis-trans isomerase activity in vitro and can play roles in protein folding and transport, RNA splicing and the regulation of multi-protein complexes in cells. In addition to enzymic activity, many immunophilins act as molecular chaperones. This property may be conferred by the isomerase domain and/or by additional domains. Recent years have seen a great increase in the number of known immunophilin genes in parasitic protozoa and helminths and in many cases their products have been characterised biochemically and their temporal and spatial expression patterns have been examined. Some of these genes represent novel types: one example is a Toxoplasma gondii gene encoding a protein with both cyclophilin and FK506-binding protein domains. Likely roles in protein folding and oligomerisation, RNA splicing and sexual differentiation have been suggested for parasite immunophilins. In addition, unexpected roles in parasite virulence (Mip FK506-binding protein of Trypanosoma cruzi) and host immuno-modulation (e.g. 18-kDa cyclophilin of T. gondii) have been established. Furthermore, in view of the potent antiparasitic activities of cyclosporins, macrolactones and non-immunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential drug targets. Investigation of the mechanisms of action of these agents may lead to the design of potent and selective antimalarial and other antiparasitic drugs. This review discusses the properties of immunophilins in parasites and the 'animal model'Caenorhabditis elegans and relates these to our understanding of the roles of these proteins in cellular biochemistry, host-parasite interaction and the antiparasitic mechanisms of the drugs that bind to them.

Amino Acid Sequence↗
PubMed2003

The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20.

Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl-prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 (thylakoid lumen PPIase of 20 kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N-termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.

Amino Acid Sequence↗
PubMed1974

Glucose phosphate isomerase deficiency with hereditary nonspherocytic hemolytic anemia.

Eight children (5 living, 3 deceased) with severe hereditary nonspherocytic hemolytic anemia caused by glucose phosphate isomerase deficiency have been observed in two Kentucky and Indiana families. All affected children were severely anemic in early life. Three deaths occurred in young patients who did not receive adequate transfusions of blood or whose parents refused to permit splenectomy. Splenectomy generally abolishes the requirement for blood transfusion. No patient has required regular transfusion of blood after puberty. Growth and development have been surprisingly normal and no patient has died of infection. The anemia is expressed as an autosomal recessive trait, but the enzyme variant can be detected in hematologically normal heterozygotes. The abnormal isomerase molecule is heat labile and is contained in neutrophils and lymphocytes as well as in erythrocytes.

Adult↗
PubMed2003

Molecular cloning and characterization of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase cDNAs from Japanese eel ovary.

3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) is a crucial steroidogenic enzyme which catalyzes an essential step in the biosynthesis of all classes of steroid hormones. Two closely related cDNAs, encoding Japanese eel ovarian types I and II 3beta-HSD, were cloned and characterized. Both cDNAs putatively encoded 375 amino acid residues sharing high sequence homology with those of rainbow trout (71%) and mammalian (approximately 45-50%) 3beta-HSD. Transient expression of types I and II 3beta-HSD in COS-7 cells revealed that both proteins possess 3beta-hydroxysteroid dehydrogenase as well as Delta(5)-Delta(4) isomerase activity for both pregnenolone and dehydroepiandrosterone, with the preference of pregnenolone over dehydroepiandrosterone as substrate, although the type I protein is more active than the type II. By northern blot analysis, a single band of the 3beta-HSD transcript of approximately 1.5kb in length was observed in ovarian tissue and the total transcript abundance of both 3beta-HSDs remained constant throughout ovarian development artificially induced by gonadotropin-rich salmon pituitary homogenate. This lack of change in 3beta-HSD transcript abundance during ovarian development did not correlate with the fluctuation of its enzymatic activity reported previously, which may suggest that changes in 3beta-HSD activity during ovarian development may be, in part, post-transcriptionally regulated in the Japanese eel ovary.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Comparisons of hydroperoxide isomerase and monooxygenase activities of cytochrome P450 for conversions of allylic hydroperoxides and alcohols to epoxyalcohols and diols: probing substrate reorientation in the active site.

Ferric cytochrome P450 (P450) catalyzes intramolecular rearrangements of hydroperoxides to diols by heterolytic scission of the O-O bond and insertion of the terminal oxygen atom into the resulting alcohol. The goals of this work were to further characterize the regio- and stereochemistry of P450 isomerase activity using allylic hydroperoxides and to compare these reactions with NADPH-supported monooxygenations of the corresponding alcohols. Microsomes from phenobarbital-treated rats or purified P450 2B1 catalyzed the conversions of several peroxyquinols, including 2-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,5-dien-1-one (BMPOOH) and its 2,6-dimethyl derivative (TMPOOH), to diols and to alpha,beta-epoxyquinols by predominant (>93%) cis addition of oxygen to the least hindered double bond. Monooxygenation of the 4-hydroxy analogues (quinols) yielded identical cis-epoxyquinols, and both isomerization and monooxygenation reactions exhibited similar enantioselectivities (32-74% enantiomer excess). Regioselectivities were similar for BMPOOH and BMPOH (epoxyquinol:diol ratios of 5.8-7.6), but quite different for TMPOOH and TMPOH (ratios of 0.6 and 6.4, respectively). Bulky peroxyquinols and quinols derived from the A-rings of 17beta-estradiol and estrone were utilized to further compare these reactions. Both estradiol derivatives underwent approximately equal amounts of 6beta-hydroxylation and 1,2-epoxidation. The estrone derivatives also underwent 6beta-hydroxylation, but only estrone quinol yielded a second product consistent with hydroxylation at position 16. The results support several conclusions. (i) Allylic hydroperoxides may be isomerized to alpha,beta-epoxyalcohols by a heterolytic O-O cleavage mechanism with high stereoselectivity. (ii) Hydroperoxide isomerization is an efficient process relative to monooxygenation. (iii) Isomerase substrates remain in proximity to the P450 oxoferryl intermediate and are rapidly captured by the oxidant. Monooxygenase substrates, on the other hand, may bind to ferric P450 in multiple orientations and undergo more extensive substrate reorientation prior to oxidative attack.

Animals↗
PubMed1967

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose.

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K(i) values being 1.37x10(-3)-1.67x10(-3)m for fructose 1-phosphate and 7.2x10(-3)-7.9x10(-3)m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K(m) values for both liver and muscle enzymes at pH7.4 and 30 degrees were 1.11x10(-4)-1.29x10(-4)m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K(m) values for glucose 6-phosphate were in the range 5.6x10(-4)-8.5x10(-4)m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.

Journal Article↗