Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ISOMERASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed1986

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

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

Carbon-Carbon Double Bond Isomerases↗
PubMed1989

Kinetic and ultraviolet spectroscopic studies of active-site mutants of delta 5-3-ketosteroid isomerase.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes the highly efficient isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-A X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E.M., Talalay, P., & Mildvan, A.S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E.M., Piro, O.E., & Sigler, P.B. (1984) J. Biol. Chem. 259, 9096-9103]. The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 10(5.6)-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4 beta-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the Y14F mutant shows a 10(4.7)-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S.F., Kawahara, F.S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed2004

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor.

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.

Bacterial Proteins↗
PubMed2005

The structure of the Candida albicans Ess1 prolyl isomerase reveals a well-ordered linker that restricts domain mobility.

Ess1 is a peptidyl-prolyl cis/trans isomerase (PPIase) that binds to the carboxy-terminal domain (CTD) of RNA polymerase II. Ess1 is thought to function by inducing conformational changes in the CTD that control the assembly of cofactor complexes on the transcription unit. Ess1 (also called Pin1) is highly conserved throughout the eukaryotic kingdom and is required for growth in some species, including the human fungal pathogen Candida albicans. Here we report the crystal structure of the C. albicansEss1 protein, determined at 1.6 A resolution. The structure reveals two domains, the WW and the isomerase domain, that have conformations essentially identical to those of human Pin1. However, the linker region that joins the two domains is quite different. In human Pin1, this linker is short and flexible, and part of it is unstructured. In contrast, the fungal Ess1 linker is highly ordered and contains a long alpha-helix. This structure results in a rigid juxtaposition of the WW and isomerase domains, in an orientation that is distinct from that observed in Pin1, and that eliminates a hydrophobic pocket between the domains that was implicated as the main substrate recognition site. These differences suggest distinct modes of interaction with long substrate molecules, such as the CTD of RNA polymerase II. We also show that C. albicans ess1(-)() mutants are attenuated for in vivo survival in mice. Together, these results suggest that CaEss1 might constitute a useful antifungal drug target, and that structural differences between the fungal and human enzymes could be exploited for drug design.

Amino Acid Sequence↗
PubMed1999

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1.

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.

Alanine↗
PubMed1980

Bovine liver thiol-protein disulphide oxidoreductases. An alternative method for differential purification and resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of 'thiol-protein disulphide oxidoreductase' by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and 'thiol-protein disulphide oxidoreductase' is discussed.

Animals↗
PubMed1981

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.

Alkynes↗
PubMed1983

Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction.

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.

Animals↗
PubMed1988

Molecular cloning of a multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit, protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. Comparison of cDNA-deduced amino acid sequences with those in other species.

Several recent studies indicate that a single polypeptide may act as the beta-subunit of prolyl 4-hydroxylase, the enzyme protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. We report here the isolation and characterization of cDNA clones encoding this multifunctional protein in the chicken. All the coding sequences were determined on the basis of nucleotide sequencing of five cDNA clones and amino acid sequencing of the N-terminal end of the chicken beta-subunit. The processed polypeptide contains 493 amino acid residues, the size of the respective mRNA being about 2.7 kb. The chicken beta-subunit cDNA sequences were 78% homologous to the previously reported human beta-subunit cDNA sequences at the nucleotide level and 85% homologous at the amino acid level. The homology of the chicken beta-subunit sequences to those reported for bovine thyroid-hormone-binding protein and rat protein disulphide-isomerase was also 85% at the amino acid level. Primary-structure comparisons between the four species indicated that the two proposed active sites of protein disulphide-isomerase, the two Trp-Cys-Gly-His-Cys-Lys sequences, are located within highly conserved regions, which are also homologous to the active sites of a number of thioredoxins. The middle of the polypeptide has an additional conserved region 100 amino acid residues in length in which the degree of homology between the four species is 94% at the amino acid level. This long conserved region may also be important for some of the multiple functions of the protein. The four extreme C-terminal amino acids of the polypeptide in all four species are Lys-Asp-Glu-Leu, a sequence that has been suggested to function as a signal for the retention of a protein in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1995

Independence of the chaperone activity of protein disulfide isomerase from its thioredoxin-like active site.

Protein disulfide isomerase (PDI) alkylated at thiols of the thioredoxin-like -CHC- active sites is devoid of isomerase activity, but its chaperone-like activity to increase the reactivation yield and prevent the aggregation of guanidine hydrochloride-denatured D-glyceraldehyde-3-phosphate dehydrogenase upon dilution is unimpaired. A peptide of 28 amino acids markedly inhibits both the enzyme and the chaperone activities of PDI. The above results indicate that the -CGHC- active site is necessary for the isomerase activity but not required for the chaperone activity of PDI, whereas the peptide binding site is essential for both activities.

Alkylation↗
PubMed1995

A novel function of Escherichia coli chaperone DnaJ. Protein-disulfide isomerase.

Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes. The DnaJ protein of Escherichia coli and the DnaJ-like proteins of eukaryotes are known as molecular chaperones and specific regulators of DnaK-like proteins and are involved in protein folding and renaturation after stress. In this study we show that DnaJ, like thioredoxin, protein-disulfide isomerase, and DsbA, possesses an active dithiol/disulfide group and catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). These results suggest that, in addition to its known function as a chaperone, DnaJ might be involved in controlling the redox state of cytoplasmic, membrane, or exported proteins.

Animals↗
PubMed1999

Characterization of PECI, a novel monofunctional Delta(3), Delta(2)-enoyl-CoA isomerase of mammalian peroxisomes.

We report here the identification and characterization of human and mouse PECI, a novel gene that encodes a monofunctional peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase. Human and mouse PECI were identified on the basis of their sequence similarity to Eci1p, a recently characterized peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae. Cloning and sequencing of the human PECI cDNA revealed the presence of a 1077-base pair open reading frame predicted to encode a 359-amino acid protein with a mass of 39.6 kDa. The corresponding mouse cDNA contains a 1074-base pair open reading frame that encodes a 358-amino acid-long protein with a deduced mass of 39.4 kDa. Northern blot analysis demonstrated human PECI mRNA is expressed in all tissues. A bacterially expressed form of human PECI catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 27 units/mg of protein. The human and mouse PECI proteins contain type-1 peroxisomal targeting signals, and human PECI was localized to peroxisomes by both subcellular fractionation and immunofluorescence microscopy techniques. The potential roles for this monofunctional Delta(3),Delta(2)-enoyl-CoA isomerase in peroxisomal metabolism are discussed.

Amino Acid Sequence↗
PubMed1999

Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

Chromatography, Affinity↗
PubMed2002

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro.

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.

Animals↗
PubMed2005

The Ess1 prolyl isomerase is dispensable for growth but required for virulence in Cryptococcus neoformans.

Cryptococcus neoformans is an important human fungal pathogen that also serves as a model for studies of fungal pathogenesis. C. neoformans contains several genes encoding peptidyl-prolyl cis/trans isomerases (PPIases), enzymes that catalyse changes in the folding and conformation of target proteins. Three distinct classes of PPIases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. This paper reports the cloning and characterization of ESS1, which is believed to be the first (and probably only) parvulin-class PPIase in C. neoformans. It is shown that ESS1 from C. neoformans is structurally and functionally homologous to ESS1 from Saccharomyces cerevisiae, which encodes an essential PPIase that interacts with RNA polymerase II and plays a role in transcription. In C. neoformans, ESS1 was found to be dispensable for growth, haploid fruiting and capsule formation. However, ESS1 was required for virulence in a murine model of cryptococcosis. Loss of virulence might have been due to the defects in melanin and urease production observed in ess1 mutants, or to defects in transcription of as-yet-unidentified virulence genes. The fact that Ess1 is not essential in C. neoformans suggests that, in this organism, some of its functions might be subsumed by other prolyl isomerases, in particular, cyclophilins Cpa1 or Cpa2. This is supported by the finding that ess1 mutants were hypersensitive to cyclosporin A. C. neoformans might therefore be a useful organism in which to investigate crosstalk among different families of prolyl isomerases.

Amino Acid Sequence↗
PubMed1981

Identification and purification of distinct isomerase and decarboxylase enzymes involved in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli.

The possible involvement of an isomerase in the 4-hydroxyphenylacetate meta-cleavage pathway has been studied. 5-Carboxymethyl-2-hydroxymuconate has been shown to undergo both spontaneous and enzyme-catalysed isomerisation to give 5-carboxymethyl-2-oxo-hex-3-ene-1,5-dioate, a compound with an absorbance maximum at 246 nm. The latter compound rather than the former is the substrate for a decarboxylase that produces 2-hydroxyhepta-2,4-diene,1,7-dioate. The isomerase and decarboxylase enzymes have been purified to over 90% homogeneity. Mg2+ is required for the decarboxylase reaction but not for the isomerase.

Carbon-Carbon Double Bond Isomerases↗
PubMed1985

Studies on the formation of intrachain disulphide bonds in newly biosynthesised bovine prolactin. Role of protein-disulphide isomerase.

Native disulphide-bonded prolactin (band III) was distinguished from reduced prolactin (band II) and intermediate unstable disulphide-linked conformations by: (a) faster mobility of the former in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and (b) high-pressure liquid chromatography analyses of tryptic-digested peptides derived from prolactin in various conformations during its refolding pathway from reduced, unfolded to native conformation. The electrophoretic separation has been used to examine the state of disulphide bonding in newly synthesised prolactin translated from bovine pituitary mRNA in a rabbit reticulocyte translation system supplemented with nuclease-treated dog pancreatic microsomal membranes. The formation of correct disulphide pairing in prolactin (band III), synthesised in the in vitro translation system in the presence of pancreatic microsomes, required the presence of a thiol oxidant such as oxidised glutathione during the translation. The action of thiol oxidants on the in vitro biosynthesised and microsomally processed prolactin were both dose-dependent and catalytic; non-thiol oxidants such as NAD+ and NADP+ were ineffective. Examination of the time course of addition of oxidised glutathione to translating lysates showed that efficient and correct disulphide pairing in newly biosynthesised prolactin occurred when the oxidant was present co-translationally, but much lower yields of correctly disulphide-bonded prolactin were obtained when the oxidant was added after translation and processing were complete. The presence of protein-disulphide isomerase in dog pancreatic microsomes, employed in the in vitro translation system to process preprolactin, was demonstrated by (a) two-dimensional polyacrylamide gel electrophoresis of the membrane proteins, and (b) enzymic activity to accelerate reactivation of scrambled ribonuclease. Protein-disulphide isomerase activity was latent in intact microsomal vesicles, full activity being expressed upon sonication. A procedure has been devised to prepare pancreatic microsomal vesicles depleted of protein-disulphide isomerase which are active in processing and segregating in vitro biosynthesised prolactin. These membranes in the presence of low concentrations of oxidised glutathione are less active but in the presence of saturating levels of oxidised glutathione are fully competent in forming correct disulphide bridges in newly synthesised prolactin.

Animals↗
PubMed1989

Cycloeucalenol-obtusifoliol isomerase. Structural requirements for transformation or binding of substrates and inhibitors.

The molecular features of 19 synthetic substrates and ground-state analogues of cycloeucalenol, the natural substrate of cycloeucalenol - obtusifoliol isomerase, a membrane-bound enzyme specific to higher plants, and of 9 synthetic carbocationic analogues of the high-energy intermediate occurring during the reaction catalyzed by the isomerase, were related to their ability to be transformed by this enzyme (catalytical competence) and their potency as inhibitors of this enzyme. With substrates and ground-state analogues it has been possible to determine at least two critical domains: significant binding requires the presence of the 3 beta-hydroxyl group on the ring A with the correct stereochemistry together with absence of a 4 beta-methyl group. Moreover initial enzyme-substrate interaction appears to be dependent upon the accessibility of the 3 beta-oxygen. Substitutions on the ring B do not preclude binding whereas they are of great influence on substrate transformation. Modifications of the ring A and other modifications suggest that ground-state and high-energy intermediate analogues bind two different conformations of the isomerase active site.

Binding Sites↗