Reaction of hemerythrin with disulfides

The reactions of hemerythrin from Phascolopsis gouldii with the specific sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoate), 2,2'-dithiodipyridine, and 4,4'-dithiodipyridine were studied at 25 degrees C. Spectrophotometric measurements showed that 1 mol of disulfide reacted per protein subunit consistent with a single cysteine at residue 50. Reaction leads to dissociation of the octameric structure of the native protein to monomers. The first-order rate constants at 25 degrees C and pH 9.0 for reactions of methemerythrin [(1.5 +/- 0.3) X 10(-3) s-1] and metazidohemerythrin [(4.0 +/- 0.3) X 10(-3) s-1] are independent of both the concentration and the nature of the disulfide. The reactions of methemerythrin are strongly inhibited by ClO4-ion, which however has no effect on the rates of those of metazidohemerythrin. The first-order kinetic behavior is ascribed to a conformational change involving the protein controlling the reaction, and this slow change appears to dominate a number of the reactions of hemerythrin.     

Intersubunit disulfides of the follitropin receptor

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.     

Sulfurization of dinucleoside phosphite triesters with chiral disulfides

Sixteen chiral analogues of phenylacetyl disulfide (PADS) and 5-methyl-3H-1,2,4-dithiazol-3-one (MEDITH) were used to sulfurize five dithymidine phosphite triesters, each incorporating a β-cyanoethoxy or siloxy group. Each mixture of S(P):R(P) phosphite triester diastereomers was combined with approximately one fourth of an equivalent of each of the sulfurizing reagents, and the R(PS):S(PS) diastereomer ratios of the resulting phosphite sulfides or phosphorothioates were determined by reverse-phase HPLC. Diastereoselectivities and corresponding diastereomeric excess (de) values were calculated by correcting for the starting triester diastereomer ratios. The highest de values for R(PS) and S(PS) phosphorothioates were 14.7% and 7.9%, respectively, both using MEDITH analogues.     

Interaction of pantetheinase with sulfhydryl reagents and disulfides

The effect of many thiol reagents and disulfides on pantetheinase (E.C. 3.5.1.-; pantetheine hydrolase) was studied in the presence or absence of S-pantetheine-3-pyruvate as substrate. Iodoacetamide, iodoacetate, bromopyruvate and N-ethylmaleimide irreversibly inactivate the enzyme at very different rates. Inactivation constants, corrected for the different reactivity of halogeno derivatives with non-protein thiols, suggest the presence of an essential sulfhydryl group in the enzyme and a negatively charged environment near this group. p-Chloromercuribenzoate is the most effective inhibitor; 2-nitro-5-thiocyanobenzoate, o-iodosobenzoate and hydrogen peroxide give a biphasic inhibition pattern, indicating the existence of two sulfhydryl groups whose modification affects activity. Organic arsenicals decrease activity to about 50%. Neutral and positively charged disulfides are effective inhibitors. Substrate protects the enzyme from inactivation, except in the case of negatively charged disulfides, where the presence of substrate enhances the inhibitory effect. Titration with Ellman's reagent or 4,4'-dithiodipyridine under various experimental conditions demonstrated the existence of two sulfhydryls and three disulfides in the fully active enzyme. Pantetheinase may become inactive during purification with concomitant loss of one titrable sulfhydryl group.     

Copper-containing oxidases

Major advances have been made during 1997 and 1998 toward understanding the structure/function relationships of the active sites in copper-containing oxidases. Central to this progress has been the elucidation of crystal structures for many of these enzymes. For example, studies of the mechanisms of biogenesis and/or catalysis of amine oxidase and galactose oxidase have been both stimulated and directed by the availability of structures for these proteins. Similarly, it is anticipated that the recently published crystal structures of peptidylglycine alpha-hydroxylating monooxygenase and laccase will contribute greatly toward understanding the roles of copper in these two proteins.     

Dual oxidases

Reactive oxygen species (ROS) have an important role in various physiological processes including host defence, mitogenesis, hormone biosynthesis, apoptosis and fertilization. Currently, the most characterized ROS-producing system operates in phagocytic cells, where ROS generated during phagocytosis act in host defence. Recently, several novel homologues of the phagocytic oxidase have been discovered and this protein family is now designated as the NOX/DUOX family of NADPH oxidases. NOX/DUOX enzymes function in a variety of tissues, including colon, kidney, thyroid gland, testis, salivary glands, airways and lymphoid organs. Importantly, members of the enzyme family are also found in non-mammalian species, including Caenorhabditis elegans and sea urchin. The physiological functions of novel NADPH oxidase enzymes are currently largely unknown. This review focuses on our current knowledge about dual oxidases.     

High torsional energy disulfides: relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent beta-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained beta-sheet, are required.     

Maize root peroxidases: relationship with polyphenol oxidases

Maize root peroxidases (POD) may also have polyphenol oxidase (PPO) activity as shown by using 3-amino-9-ethylcarbazole or DOPA as hydrogen donor to detect isoenzymes after disc gel electrophoresis. Copper chelators inhibited POD activity, and since PODs are haemoproteins, it can be concluded that copper chelators are not entirely specific for Cu enzymes. This raises the question whether PPO are only Cu enzymes. In POD preparations contaminated by catalase, POD activity could be over-estimated; this could be due to the auto-oxidation of the hydrogen donor or to stimulation of PPO activity by oxygen, as demonstrated with DOPA, dopamine and gallic acid. No correlation was found between the chemical nature of the substrate and the type of peroxidatic or oxidatic oxidation.     

Enzyme regulation by biological disulfides

More than a dozen enzymes have been found to be activated or inhibitedin vitro by disulfide-exchange between the protein and small-molecule disulfides. Accordingly, thiol/disulfide ratio changesin vivo may be of great importance in the regulation of cellular metabolism. An awareness of this regulatory mechanism in both host cells and parasites, coupled with information on the presence or absence of key enzymes, may lead to rational drug design against certain diseases involving thiol intermediates, including trypanosomiasis.Abbreviations GSSG glutathione disulfide - CoASSG mixed disulfide of coenzyme A and glutathione - CoASSCoA coenzyme A disulfide - PrSSG protein mixed disulfide - Cystamine 2,2-dithiobioethanamine [ aminoethyl] disulfide - GSSO₂G Glutathione thiosulfonate - PFK Phosphofructokinase - FBPase Fructose 1,6-bisphosphatase     

Multiple ways to make disulfides

Our concept of how disulfides form in proteins entering the secretory pathway has changed dramatically in recent years. The discovery of endoplasmic reticulum (ER) oxidoreductin 1 (ERO1) was followed by the demonstration that this enzyme couples oxygen reduction to de novo formation of disulfides. However, mammals deficient in ERO1 survive and form disulfides, which suggests the presence of alternative pathways. It has recently been shown that peroxiredoxin 4 is involved in peroxide removal and disulfide formation. Other less well-characterized pathways involving quiescin sulfhydryl oxidase, ER-localized protein disulfide isomerase peroxidases and vitamin K epoxide reductase might all contribute to disulfide formation. Here we discuss these various pathways for disulfide formation in the mammalian ER and highlight the central role played by glutathione in regulating this process.     

Antivitamin activity of O-acyloxythiamine disulfides

B1-antivitamin activity of symmetrical oxythiamine disulphide esters with succinic and o-phthalic acids has been studied in experiments on albino mice. It is shown that O-acyloxythiamine disulphides exert more profound and prolonged inhibitory effect on the transketolase activity in the animal body in comparison with the known antagonist of vitamin B1, oxythiamine, and the initial oxythiamine disulphide.