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At least 37 records · Page 2Linked to original sources
PubMed1992

Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. This multifunctional polypeptide has two independently acting catalytic sites for the isomerase activity.

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.

Amino Acid Sequence↗
PubMed1995

Protein disulphide isomerase and a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase are in transient contact with secretory proteins during late stages of translocation.

The transport of a presecretory protein into the mammalian endoplasmic reticulum can be divided into early translocation events which include specific targeting of the presecretory protein to and insertion into the endoplasmic reticulum membrane and late translocation events, comprising signal sequence cleavage, completion of translocation and folding of the secretory protein into a functional conformation. The microsomal membrane proteins Sec61 alpha p and translocating-chain-associating membrane protein were previously identified as being in close contact with a nascent presecretory protein at an early step of translocation. Here, we investigated whether additional microsomal proteins are in contact with translocating chains during or immediately after transit. This was addressed by crosslinking after release of the nascent chain from Sec61 alpha p. We observed two additional membrane proteins interacting with the nascent precursor in the early stages of translocation and three lumenal proteins interacting with the processed polypeptide chain in the late stages of translocation. One of the lumenal proteins was identified as protein disulphide isomerase by immunoprecipitation. Another of the lumenal proteins was suggested to be a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase by the effect of cyclosporin A. We propose that molecular chaperones, such as protein disulphide isomerase and cyclophilin may represent two of the lumenal proteins which are involved in completion of translocation.

Amino Acid Isomerases↗
PubMed1981

Comparative inactivation and inhibition of the anomerase and isomerase activities of phosphoglucose isomerase.

Several metabolic compounds have been found to be competitive inhibitors of the anomerase activity of phosphoglucose isomerase (EC 5.3.1.9).Ki values for erythrose 4-phosphate, 6-phosphogluconate, and fructose 1,6-bisphosphate for the anomerase reaction are 0.32 muM, 21 muM, and 84 muM respectively at 0 degree and pH 8.2. A significant difference between the fructose 1,6-bisphosphate inhibition constants for both activities was found (Ki(isomerase) = 800 muM and Ki(anomerase) = 140 muM). Also the Km values for both activities were found to be significantly different (Km(isomerase) = 140 muM and Km(anomerase) = 3.6 muM). Attempts to independently alter the anomerase to isomerase activity ratio through protein modification yielded mixed results. While several modifying reagents destroyed the catalytic activities at identical rates, inactivation by iodoacetamide or pyridoxal 5' phosphate sensitized photo-oxidation displayed differential initial effects on the two activities with the anomerase activity being the less affected. These data support the theory that an imidazole residue is catalytically important for isomerization, but less so for anomerization.

Animals↗
PubMed1991

Evidence for distinct dehydrogenase and isomerase sites within a single 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein.

Complementary DNA encoding human 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3 beta-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3 beta-HSD protein. In addition, using 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta, 17 beta-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

Androgen Antagonists↗
PubMed1998

Protein disulfide isomerase assists protein folding as both an isomerase and a chaperone.

Protein disulfide isomerase (PDI) is the physiological catalyst of native disulfide bond formation of nascent peptides in the cells. As a foldase, PDI has both isomerase and chaperone activities. The chaperone activity is intrinsic and independent of its isomerase activity. Both chaperone and isomerase activities are required for PDI to assist folding of denatured and reduced disulfide-containing proteins. PDI may have great applications in protein production by bioengineering for its function as a foldase.

Disulfides↗
PubMed1994

cDNA cloning and amino acid sequence of human mitochondrial delta 3 delta 2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase.

We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735. The human mitochondrial isomerase exhibits a 74% (78%) sequence identity with the corresponding rat counterpart at amino acid (nucleotide) level(s). Many basic amino acid residues in rat isomerase have been changed to acidic or neutral residues in human enzyme, explaining the differences observed between these proteins.

Amino Acid Sequence↗
PubMed1991

Amino acid sequence similarities of the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase and peroxisomal multifunctional delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase enzyme in rat liver. The proposed occurrence of isomerization and hydration in the same catalytic domain of the multifunctional enzyme.

We report the isolation and characterization of a cDNA encoding the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase from rat liver. Tryptic fragments of the purified protein were generated, purified, and sequenced. A rat liver cDNA library, constructed in the plasmid vector pUEX1 was screened with oligonucleotides synthesized on the basis of peptide sequences. The obtained clone contained 783 bases predicting to code the entire mature protein of 261 amino acids. The molecular weight of 29,300 predicted from cDNA-derived sequences was consistent with the subunit size determined earlier. A high degree of similarity was noted between the amino acid sequence of isomerase and that of the amino-terminal half of peroxisomal multifunctional isomerase-hydratase-dehydrogenase enzyme and mitochondrial 2-enoyl-CoA hydratase in rat liver. These similarities also appeared at the level of predicted secondary structural elements, suggesting that hte rat multifunctional enzyme has both the isomerization and hydration activities in the amino-terminal domain. This idea is further supported by the proposed existence of only one CoA-binding site in the amino-terminal half of the multifunctional enzyme and by previous studies suggesting that the transfer of the substrate from the isomerization site to the hydration site occurs without aqueous bulk phase (Palosaari P.M., and Hiltunen, J. K. (1990) J. Biol. Chem. 265, 2446-2449).

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1984

Stimulation of mouse liver corticosteroid side chain isomerase by cobaltous and nickelous ions: evidence for an endogenous inhibitor of isomerase activity.

Corticosteroid side chain isomerase of mouse liver cytosol was stimulated by Co2+ and Ni2+. The magnitude of stimulation increased with incubation time. For Co2+ and Ni2+, respective enhancements were 2.8- and 4.0-fold at 15 min and 3.9- and 5.0-fold at 60 min. The relationship between steroid substrate concentration (11-deoxy-[21-3H]corticosterone) and initial velocity was consistent with a model in which the cations reacted with a cytosol inhibitor of isomerase activity. Enzyme, partially purified by ammonium sulfate fractionation and gel filtration, had a 6.8-fold increased specific activity. Co2+ and Ni2+ enhanced the activity of partially purified enzyme 1.6- and 1.9-fold. Unlike the cytosol, stimulation was achieved without lag and was not altered by prolonged incubation. Metal ion chelating agents did not have a consistent effect on the activity of the partially purified enzyme. Cyanide and alpha,alpha-dipyridyl increased, and dithizone and 8-hydroxyquinoline decreased activity. The data are not consistent with the hypothesis that side chain isomerase is a metalloenzyme. It is concluded that Co2+ and Ni2+ stimulate the enzyme by removing an endogenous inhibitor.

Animals↗
PubMed1996

The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.

The microsomal triacylglycerol transfer protein (MTP), an alpha beta dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The beta subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the alpha subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the alpha and beta subunits were co-expressed, soluble alpha beta dimers were formed with significant triacylglycerol transfer activity. Expression of the alpha subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded alpha beta dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the beta subunit in MTP seems to be to keep the alpha subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the beta subunit is not required for this function.

Animals↗
PubMed1997

Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.

Agkistrodon↗
PubMed1980

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase. Sequence homology with triosephosphate isomerase.

N-Bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pKa values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivty methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

Affinity Labels↗
PubMed2004

Replacement of domain b of human protein disulfide isomerase-related protein with domain b' of human protein disulfide isomerase dramatically increases its chaperone activity.

We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity [J. Biol. Chem. 279 (2004) 4604]. To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants. Interestingly, when the b domain of hPDIR was replaced with the b' domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed. However, this mutant showed decreased oxidative refolding of alpha1-antitrypsin. The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity. These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity.

Amino Acid Motifs↗
PubMed1990

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8.

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.

Aldose-Ketose Isomerases↗
PubMed1992

Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase.

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.

Amino Acid Isomerases↗
PubMed1981

A simple procedure to obtain yeast hexokinase free of glucosephosphate isomerase and mannosephosphate isomerase.

A hexokinase preparation was obtained from a Saccharomyces cerevisiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate. The supernatant fraction corresponding to 40-60 % saturation showed the lowest content in GPI and MPI activity. The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose. The results were similar to those obtained with pure commercial hexokinase.

Ammonium Sulfate↗
PubMed1995

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell.

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

Base Sequence↗
PubMed2003

Bifunctional phosphoglucose/phosphomannose isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum constitute a novel enzyme family within the phosphoglucose isomerase superfamily.

The hyperthermophilic crenarchaeon Aeropyrum pernix contains phosphoglucose isomerase (PGI) activity. However, obvious homologs with significant identity to known PGIs could not be identified in the sequenced genome of this organism. The PGI activity from A. pernix was purified and characterized. Kinetic analysis revealed that, unlike all known PGIs, the enzyme catalyzed reversible isomerization not only of glucose 6-phosphate but also of epimeric mannose 6-phosphate at similar catalytic efficiency, thus defining the protein as bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). The gene pgi/pmi encoding PGI/PMI (open reading frame APE0768) was identified by matrix-assisted laser desorption ionization time-of-flight analyses; the gene was overexpressed in Escherichia coli as functional PGI/PMI. Putative PGI/PMI homologs were identified in several (hyper)thermophilic archaea and two bacteria. The homolog from Thermoplasma acidophilum (Ta1419) was overexpressed in E. coli, and the recombinant enzyme was characterized as bifunctional PGI/PMI. PGI/PMIs showed low sequence identity to the PGI superfamily and formed a distinct phylogenetic cluster. However, secondary structure predictions and the presence of several conserved amino acids potentially involved in catalysis indicate some structural and functional similarity to the PGI superfamily. Thus, we propose that bifunctional PGI/PMI constitutes a novel protein family within the PGI superfamily.

Aeropyrum↗
PubMed2004

A novel phosphoglucose isomerase (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A resolution.

The crystal structure of a dual specificity phosphoglucose isomerase (PGI)/phosphomannose isomerase from Pyrobaculum aerophilum (PaPGI/PMI) has been determined in native form at 1.16-A resolution and in complex with the enzyme inhibitor 5-phosphoarabinonate at 1.45-A resolution. The similarity of its fold, with the inner core structure of PGIs from eubacterial and eukaryotic sources, confirms this enzyme as a member of the PGI superfamily. The almost total conservation of amino acids in the active site, including the glutamate base catalyst, shows that PaPGI/PMI uses the same catalytic mechanisms for both ring opening and isomerization for the interconversion of glucose 6-phosphate (Glc-6-P) to fructose 6-phosphate (Fru-6-P). The lack of structural differences between native and inhibitor-bound enzymes suggests this activity occurs without any of the conformational changes that are the hallmark of the well characterized PGI family. The lack of a suitable second base in the active site of PaPGI/PMI argues against a PMI mechanism involving a trans-enediol intermediate. Instead, PMI activity may be the result of additional space in the active site imparted by a threonine, in place of a glutamine in other PGI enzymes, which could permit rotation of the C-2-C-3 bond of mannose 6-phosphate.

Amino Acid Sequence↗