Characterisation and quantitation of a selenol intermediate in the reaction of ebselen with thiols.

The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.     

Structural evidence for a common intermediate in small G protein-GEF reactions

Rho of plants (Rop) proteins belong to the superfamily of small GTP-binding (G) proteins and are vital regulators of signal transduction in plants. In order to become activated, Rop proteins need to exchange GDP for GTP, an intrinsically slow process catalyzed by guanine nucleotide exchange factors (GEFs). RopGEFs show no homology to animal RhoGEFs, and the catalytic mechanism remains elusive. GEF-catalysed nucleotide exchange proceeds via transient ternary and stable binary complexes. While a number of structural studies have analyzed binary nucleotide-free G protein-GEF complexes, very little is known about the ternary complexes. Here we report the X-ray structure of the catalytic PRONE domain of RopGEF8 from Arabidopsis thaliana, both alone and in a ternary complex with Rop4 and GDP. The features of the latter complex, a transient intermediate of the exchange reaction never directly observed before, suggest a common mechanism of catalyzed nucleotide exchange applicable to small G proteins in general.     

The reactions of mercurated pyrimidine nucleotides with thiols and with hydrogen sulfide.

In the presence of thiols, 5-mercuripyrimidine nucleotides are quantitatively converted to 5-thiomercuri derivatives, but these compounds are unstable and decompose at a rate dependent on the nature of the thiol. The decomposition involves three different reactions and proceeds via a symmetrical mercury derivative of the nucleotide. The end product is the unmodified nucleotide. Similar reactions occur in the presence of hydrogen sulfide. Since mercurated nucleoside triphosphates are substrates for RNA- and DNA polymerase only in the form of thiomercuri derivatives, this implies that when DNA is replicated or transcribed in vitro with a mercurated substrate, the latter is rapidly demercurated to the unmodified substrate which is incorporated as well. Hence the product of the in vitro synthesis can only be partially mercurated in any one pyrimidine. Also, formation of cross-links in the resulting polymer is possible.     

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.     

On the reaction of iron bleomycin with thiols and oxygen.

Iron(III)bleomycin undergoes a redox reaction with thiols. Evidence from epr and UV-visible absorbance spectra indicate that the metal complex forms an intermediate with sulfhydryl groups presumably by adding a sixth ligand. In the presence of oxygen iron bleomycin cycles between its two oxidation states to catalyze the generation of oxygen free radicals using thiols as a source of electrons.     

Formation of an aspartyl phosphate intermediate in the reactions of nucleoside phosphotransferase from carrots

The nucleoside phosphotransferase from carrots forms N-phosphorylhydroxylamine when substrates are hydrolysed in the presence of hydroxylamine. Denaturation of the enzyme after short incubation with the substrates leads to a protein, in which, after reduction with [3H]NaCNBH3 and complete hydrolysis with 6 M HCl, labelled homoserine can be detected. The first experiment provides evidence for an activated phosphorylenzyme, the second experiment shows that the intermediate is an acyl phosphate formed by a nucleophilic attack of an aspartate beta-carboxylate group.     

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

Endo-β-N-acetylglucosaminidase H (endo H) is an enzyme which acts on asparagine- and lipid-linked oligosaccharides containing five or more mannose residues. Complex oligosaccharides and glycopeptides are completely resistant to the action of the enzyme. We have carried out pulse-chase experiments with ³⁵S-methionine and ³H-mannose in uninfected cells and in cells infected with Sindbis virus and vesicular stomatitis virus (VSV). In each case, the labeled materials were analyzed for sensitivity to endo H by polyacrylamide gel electrophoresis and gel filtration. We find that endo H releases all the labeled mannose from pulse-labeled proteins. Initially, the released material is nearly identical in size to the endo H cleavage product derived from lipid-linked oligosaccharides present in the same cells. During chase periods, ³⁵S-methionine and ³H-mannose protein becomes increasingly resistant to the enzyme. Moreover, the ³H-mannose-labeled material released from the protein during chase periods is smaller in size than the oligosaccharide from the lipid.On the basis of these results and results from other laboratories, we propose that during glycosylation of asparagine residues, a common oligosaccharide is transferred from the lipid carrier to protein and is subsequently processed to yield the so-called “high mannose” and “complex” oligosaccharides. Since, on the basis of present evidence, the lipid-linked oligosaccharide contains two N-acetylglucosamine, 8–12 mannose and 1–2 glucose molecules, it seems probable that the carbohydrate-processing systems remove half or more of the mannose and all of the glucose residues at sites destined to become complex glycopeptides. Removal of mannose and glucose residues may also occur at sites destined to become mature high mannose glycopeptides.     

Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.     

On the modification of adenosine kinase by thiols.

The catalytic activity of adenosine kinase (EC 2.7.1.20) from yeast is very labile. Even incubation with thiols provokes a loss of two thirds of its enzymatic activity. Concomitantly, two SH-groups appear on the enzyme in addition to the single SH-group already present in the untreated enzyme, the latter being absolutely essential for activity. Treatment of adenosine kinase with thiols does not substantially affect the binding of the substrates adenosine and ATP-Mg2. The reactivity of the two newly formed SH-groups is diminished in the presence of ATP-Mg2, whereas adenosine has no influence. The opposite holds for the reactivity of the single SH-group essential for enzymatic activity. Complete reactivation of the enzymatic activity after incubation of adenosine kinase with thiols can be achieved by reoxidation of the enzyme in presence of high concentrations of adenosine. These observations suggest the notion that adenosine kinase contains an essential SH-group close to the adenosine-binding site and a disulfide bridge near to the binding site of ATP-Mg2, the latter being easily accesible to the reduction by thiols.     

Formation of the "peroxy" intermediate in cytochrome c oxidase is associated with internal proton/hydrogen transfer

When dioxygen is reduced to water by cytochrome c oxidase a sequence of oxygen intermediates are formed at the reaction site. One of these intermediates is called the "peroxy" (P) intermediate. It can be formed by reacting the two-electron reduced (mixed-valence) cytochrome c oxidase with dioxygen (called P(m)), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen (called P(r)). In recent years, evidence has accumulated to suggest that the O-O bond is cleaved in the P intermediate and that the heme a(3) iron is in the oxo-ferryl state. In this study, we have investigated the kinetic and thermodynamic parameters for formation of P(m) and P(r), respectively, in the Rhodobacter sphaeroides enzyme. The rate constants and activation energies for the formation of the P(r) and P(m) intermediates were 1.4 x 10(4) s(-1) ( approximately 20 kJ/mol) and 3 x 10(3) s(-1) ( approximately 24 kJ/mol), respectively. The formation rates of both P intermediates were independent of pH in the range 6.5-9, and there was no proton uptake from solution during P formation. Nevertheless, formation of both P(m) and P(r) were slowed by a factor of 1.4-1.9 in D(2)O, which suggests that transfer of an internal proton or hydrogen atom is involved in the rate-limiting step of P formation. We discuss the origin of the difference in the formation rates of the P(m) and P(r) intermediates, the formation mechanisms of P(m)/P(r), and the involvement of these intermediates in proton pumping.     

Formation of S-nitrosothiols from regiospecific reaction of thiols with N-nitrosotryptophan derivatives

S-nitrosothiols transport nitric oxide in vivo, and so-called transnitrosation reactions (i.e. the transfer of the nitroso function from nitrosothiol to thiolate) are believed to be involved in this process. In the present study we examined the N-nitrosotryptophan derivative-dependent nitrosation of thiols, a hitherto ignored possibility for the formation of S-nitrosothiols. The corresponding products were identified by (15)N-NMR spectrometry. The fact that the reaction proceeded under hypoxic conditions as well as in non-aqueous solution strongly indicated the occurrence of a transnitrosation reaction. Interestingly, S-nitrosothiols could only very slowly transnitrosate N-terminal-blocked tryptophan derivatives like melatonin in non-aqueous solution but did not induce such a reaction in water. The indole moiety of the N-nitrosotryptophan derivatives was fully restituted during the reaction with thiols, as demonstrated by both capillary zone electrophoresis and fluorescence spectroscopy. A determination of the Arrhenius parameters demonstrated that the corresponding rate constants were comparable with the ones known for the transfer of the nitroso function from nitrosothiol to thiolate. Thus, N-nitrosotryptophan-dependent nitrosation of thiols may occur in vivo and might offer the possibility of developing a new class of vasodilative drugs.