Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.     

Kinetics of phenolic polymerization catalyzed by peroxidase in organic media

Phenolic polymerization was carried out by enzymatic catalysis in organic media, and its kinetics was studied by using high-pressure liquid chromatography (HPLC). Phenols and aromatic amines with electron-withdrawing groups could hardly be polymerized by HRP catalysis, but phenols and aromatic amines with electron-donating groups could easily be polymerized. The reaction rate of either the para-substituted substrate or meta-substituted substrate was higher than that of ortho-substituted substrate. When ortho-position of hydroxy group of phenols was occupied by an electron-donating group and if another electron-donating group occupied para-position of hydroxy group, the reaction rate increased. Horseradish peroxidase and lactoperoxidase could easily catalyze the polymerization, but chloroperoxidase and laccase failed to yield polymers. Metallic ions such as Mn(2+), Fe(2+), or Fe(3+), and Cu(2+) could poison horseradish peroxidase to various extents, but ions such as Co(2+), Cd(2+), Zn(2+), and K(+) were not found to inhibit the reaction. (c) 1995 John Wiley & Sons, Inc.     

Differential effects of methylmercuric chloride and mercuric chloride on oxidation and iodination reactions catalyzed by thyroid peroxidase

Thyroid peroxidase (TPO), the major enzyme in the thyroid hormone synthesis, multifunctionally catalyzes (1) iodide oxidation, (2) iodination of the precursor protein, and (3) a coupling reaction of iodotyrosyl residues. The present study was carried out to examine the mercurial effects on the iodination, the second step of TPO. Purified porcine thyroglobulin or bovine serum albumin as acceptor protein was iodinated with [125I]NaI and H2O2 by purified porcine TPO. Iodinated protein was separated by acid precipitation on membrane filter or paper chromatography. Both CH3HgCl and HgCl2 dose-dependently inhibited the iodination, but HgCl2 was more potent to inhibit the iodination than CH3HgCl. These mercurial effects on the second step resemble the effects on the third step which were already reported; but are in marked contrast to the effects on the first step, where TPO was inhibited by HgCl2 but never by CH3HgCl.     

Enzymatic formation of carbohydrate rings catalyzed by single-walled carbon nanotubes

Macrocyclic carbohydrate rings were formed via enzymatic reactions around single-walled carbon nanotubes (SWNTs) as a catalyst. Cyclodextrin glucanotransferase, starch substrate and SWNTs were reacted in buffer solution to yield cyclodextrin (CD) rings wrapped around individual SWNTs. Atomic force microscopy showed the resulting complexes to be rings of 12–50 nm in diameter, which were highly soluble and dispersed in aqueous solution. They were further characterized by Raman and Fourier transform infrared spectroscopy and molecular simulation using density functional theory calculation. In the absence of SWNT, hydrogen bonding between glucose units determines the structure of maltose (the precursor of CD) and produces the curvature along the glucose chain. Wrapping SWNT along the short axis was preferred with curvature in the presence of SWNTs and with the hydrophobic interactions between the SWNTs and CD molecules. This synthetic approach may be useful for the functionalization of carbon nanotubes for development of nanostructures.     

Kinetics of the oxidation of p-aminobenzoic acid catalyzed by horseradish peroxidase compounds I and II.

The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.     

About insulin iodination: I. Monoidoination of insulin by horseradish peroxidase

Beef insulin has been iodinated according to an enzymic method. Labeled molecules have been separated quantitatively from the unlabeled ones on Ampholines. Results supplied by qualitative and quantitative studies of the distribution of iodine in the different tyrosyl residues showed that insulin was essentially iodinated on the tyrosyl residue A₁₉.     

Absorption of horse-radish peroxidase by the mucosal cells of the duodenum of mouse

Summary Horse-radish peroxidase was injected intraperitonealy to fasted and fed newborns. The protein was rapidly absorbed by the fasting animals and more slowly by the fed newborns. The tracer was progressively segregated into the supranuclear cluster of dense tubules and dense bodies. The cellular sap of several cells was deeply stained after the intraperitoneal injection. Control experiments showed that peroxidase has a high affinity for the matrix of the dense bodies in the first days of neonatal life.This work was supported by grants no. MA-3994 Medical Research Council of Canada, and by no. 9340-06 National Defense Board.The author is greatly indebted to Mr. Michel Couture for his skillful technical aid.     

Subcellular localization and polymorphism of peroxidase in horse-radish tumour and teratoma tissue

The localization of peroxidase in cells of horse-radish (Armoracia lapathifolia Gilib.) tumour and teratoma tissues was studied. Both tissue lines were derived from the same primary crown-gall tumour induced on the leaf fragments by a wild type of Agrobacterium tumefaciens B6S3. Enzymatic activity was measured in cell walls, high-density heterogeneous membrane fraction, microsomal and soluble (no particulate) fractions. The subcellular localization of enzymatic activity was distinct for each transformed tissue. Both tumour and teratoma showed similar isoenzyme patterns, but one soluble acidic isoperoxidase could be considered as a marker of cell differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enzymatic alpha-glucosaminylation of maltooligosaccharides catalyzed by phosphorylase

This paper describes phosphorylase-catalyzed enzymatic alpha-glucosaminylation for the direct incorporation of a 2-amino-2-deoxy-alpha-d-glucopyranose unit into maltooligosaccharides. When the reaction of 2-amino-2-deoxy-alpha-d-glucopyranosyl 1-phosphate as the glycosyl donor with maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, glucosaminylated oligosaccharides were produced, which were characterized by MALDI-TOF MS measurement after N-acetylation of the crude products. The N-acetylated derivative of the main product in this system was isolated by using HPLC, and its structure was confirmed by MS and (1)H NMR spectra. Furthermore, glucoamylase-catalyzed reaction of the isolated compound provided support that the alpha-glucosamine unit is positioned at the non-reducing end of the oligosaccharide.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

Enzymatic radioiodination of phospholipids catalyzed by lactoperoxidase

Phospholipids were iodinated with iodide by a lactoperoxidase-catalyzed reaction in the presence of controlled amounts of H₂O₂ which were continuously supplied by glucose oxidase + glucose. Different molecular and ionic species of inorganic iodine present in the reaction mixture (i.e., I^?, I₂, I₃^?) were eliminated by thiosulfate reduction to I^? followed by gel filtration on Sephadex LH-20 which separated I^? from the phospholipids completely. Final separation and identification of individual phospholipids were done on a column of silica gel H using a single solvent mixture consisting of CHCl₃:CH₃OH:CH₃COOH:H₂O (25:15:4:2, by volume). Application of phospholipases A₂ and D or transesterification provided evidence to indicate a covalent iodination of the fatty acid moiety of the lipids by the enzymatic process, which apparently is substitution but could also proceed by addition to the double bonds, when present.