Retinene isomerase

Rhodopsin is formed by the condensation of opsin with a cis isomer of retinene, called neo-b. The bleaching of rhodopsin releases all-trans retinene which must be isomerized back to neo-b in order for rhodopsin to regenerate. Both retinene isomers are in equilibrium with the corresponding isomers of vitamin A, through the alcohol dehydrogenase system. An enzyme is found in cattle retinas and frog pigment layers which catalyzes the interconversion of all-trans and neo-b retinene. We call it "retinene isomerase." It is soluble in neutral phosphate buffer, and precipitates between 20 and 35 per cent saturation with ammonium sulfate. In the dark, the isomerase converts all-trans and neo-b retinene to an equilibrium mixture of 5 parts neo-b and 95 parts all-trans. With opsin present to trap neo-b, the isomerase catalyzes the synthesis of rhodopsin from all-trans retinene. This reaction, however, is too slow to account for dark adaptation. Retinene is isomerized by light, but too slowly to supply the retina with neo-b. In aqueous solution the pseudoequilibrium mixture contains about 15 per cent neo-b. When all-trans retinene is irradiated in the presence of isomerase, the rate of formation of neo-b is increased about 5 times, and the pseudoequilibrium shifted so that the mixture now contains about 32 per cent neo-b. The isomerase is specific for all-trans and neo-b retinene. It does not act on two other cis isomers of retinene, nor on all-trans or neo-b vitamin A. The role of the isomerase in vision appears to be as follows: in the light, as rhodopsin is bleached to opsin and all-trans retinene, the latter is in part converted to the neo-b isomer and stored in the pigment epithelium as neo-b vitamin A. During dark adaptation, the dominant process is the trapping by opsin of neo-b retinene supplied from stores of neo-b vitamin A, and the slow isomerase-catalyzed "dark" conversion of all-trans to neo-b retinene.     

Peptidyl-prolyl isomerase inhibitors

Designed peptidyl-prolyl isomerase (PPIase) inhibitors of Pin1, cyclophilin (CyP), and FK506 binding protein (FKBP) are reviewed. Emphasis is placed on the design, structure, and biological activity of the inhibitors. While CyP and FKBP inhibitors have been explored fairly thoroughly, inhibitors of the relatively new Pin1 cell cycle regulator are in their infancy. Ligands designed for Pin1 and CyP have primarily been ground state analogues: alkenes and bicyclic compounds. For FKBP, more of the focus has been on analogues of bonds at the reactive center, the prolyl amide, because of the idea that the alpha-ketoamide of FK506 is an analogue of the twisted amide in the transition state.     

Protein disulfide isomerase

During the maturation of extracellular proteins, disulfide bonds that chemically cross-link specific cysteines are often added to stabilize a protein or to join it covalently to other proteins. Disulfide formation, which requires a change in the covalent structure of the protein, occurs as the protein folds into its three-dimensional structure. In the eukaryotic endoplasmic reticulum and in the bacterial periplasm, an elaborate system of chaperones and folding catalysts ensure that disulfides connect the proper cysteines and that the folding protein does not make improper interactions. This review focuses specifically on one of these folding assistants, protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide formation and isomerization and a chaperone that inhibits aggregation.     

Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase

Isomaltulose is a natural isomer of sucrose. It is widely used as a functional sweetener with promising properties, including slower digestion, lower glycemic index, prolonged energy release, lower insulin reaction, and less cariogenicity. It has been approved as a safe sucrose substitute by the Food and Drug Administration of the US; Ministry of Health, Labor and Welfare of Japan; and the Commission of the European Communities. This article presents a review of recent studies on the properties, physiological effects, and food application of isomaltulose. In addition, the biochemical properties of sucrose isomerases producing isomaltulose are compared; the heterologous expression, fermentation optimization, structural determination, and catalysis mechanism of sucrose isomerase are reviewed; and the biotechnological production of isomaltulose from sucrose is summarized.     

Immobilization of glucose isomerase

The immobilization of glucose isomerase (D-xylose ketol isomerase, EC 5.3.1.5) by covalently bonding to various carriers and by adsorption to ion exchange resins was attempted in order to obtain a stable immobilized enzyme which can be used for continuous isomerization of glucose in a column. Of the covalent bonding methods, the colloidal silica-glutaraldehyde method showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Ludox HS-30 bound glucose isomerase column showed a half-life of 24 days and an enzyme usage of 0.07 units per gram of isomerized sugar (d.s, fructose 45%). Of the resins used, the macromolecular type or porous type strongly basic anion exchange resins showed the highest binding capacity and gave the most stable immobilized glucose isomerase. The Amberlite IRA-904 resine-bound glucose isomerase showed a half-life of 23 days and an enzyme usage of 0.06 units per gram of isomerized sugar (d.s., fructose 45%). Based on the ease of the immobilization process, the possibility of carrier reuse and the extensive use already achieved by ion exchange resins in the sugar industry, IRA-904 resin was selected as the candidate for commercialization.     

Glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.     

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

Summary Several metabolic compounds have been found to be competitive inhibitors of the anomerase activity of phosphoglucose isomerase (EC 5.3.1.9).K_i values for erythrose 4-phosphate, 6-phosphogluconate, and fructose 1,6-bisphosphate for the anomerase reaction are 0.32 μM, 21 μM, and 84 μM respectively at 0° and pH 8.2. A significant difference between the fructose, 1,6-bisphosphate inhibition constants for both activities was found (k_i(isomerase)=800 μM and K_i(anomerase)=84 μM). Also the K_m values for both activities were found to be significantly different (K_m(isomerase)=140 μM and K_m(anomerase)=3.6 μM). 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.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Glucosephosphate isomerase polymorphism in sheep

Two types of glucosephosphate isomerase (GPI) are described for the first time in sheep. Type T produced a 3-band pattern on polyacrylamide gel electrophoresis of serum samples, whereas type M produced a pattern of 5-7 bands. When 23 Scottish Halfbred ewes showing type T were mated to 3 Suffolk rams showing type M, their 32 offspring had frequencies of 0.72 and 0.28 for T and M respectively. There was no conclusive evidence that the types were controlled by codominant alleles or by simple Mendelian inheritance and neither was sex-linked. The frequency of the rarer type (M) was sufficiently high to provide conclusive evidence of true enzyme polymorphism.     

Intracellular distribution of hydroperoxide isomerase

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.     

Isopentenyl pyrophosphate isomerase from liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6.0 and is activated by Mn(2+) in preference to Mg(2+). The K(m) value for isopentenyl pyrophosphate is 4x10(-6)m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.     

Phosphoglucose isomerase in developing teeth

Summary The distribution of phosphoglucose isomerase in developing teeth of rats, using an indirect tetrazolium technique, is described. The demonstration of this enzyme, which occupies a position of considerable importance in carbohydrate metabolism, provides additional information regarding the biochemistry of odontogenic cells.     

Autodegradation of protein disulfide isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.