Radical rearrangement and transfer reactions in proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 87-96
Author(s):  
Christian Schöneich
Keyword(s):  
Organic Chemistry ◽  
Protein Sequence ◽  
Transfer Reactions ◽  
Reaction Products ◽  
Chemical Modifications ◽  
Structure And Dynamics ◽  
Radical Rearrangement ◽  
Unexpected Reaction

Abstract Radical rearrangement and transfer reactions play an important role in the chemical modifications of proteins in vivo and in vitro. These reactions depend on protein sequence, as well as structure and dynamics. Frequently, these reactions have well-defined precedents in the organic chemistry literature, but their occurrence in proteins provides a stage for a number of novel and, perhaps, unexpected reaction products. This essay will provide an overview over a few representative examples of radical rearrangement and transfer reactions.

scholarly journals In vivo mucoadhesive strength appraisal of gum Manilkara zapota

2015 ◽  
Vol 51 (3) ◽  
pp. 689-698 ◽  
Author(s):  
Singh Sudarshan ◽  
Bothara Sunil B
Keyword(s):  
Drug Delivery Systems ◽  
Guar Gum ◽  
Methyl Cellulose ◽  
Synthetic Polymers ◽  
Chemical Modifications ◽  
Experimental Conditions ◽  
Manilkara Zapota ◽  
Seed Mucilage

The mucilage (MMZ) extracted from the seeds of Manilkara zapota(Linn.) P. Royen syn. using maceration techniques was evaluated for mucoadhesive strength by various in vitro and in vivo methods. The result showed that mucoadhesive strength of seeds mucilage have comparable property toward natural and synthetic polymers such as Guar Gum and hydroxyl propyl methyl cellulose (HPMC E5LV) under the experimental conditions used in this study. Briefly, it could be concluded that the seed mucilage of Manilkara zapota can be used as a pharmaceutical excipient in oral mucoadhesive drug delivery systems. Further, it may be appropriate to study the changes in these properties after chemical modifications.

scholarly journals In vitro and in vivo properties of therapeutic oligonucleotides containing non-chiral 3′ and 5′ thiophosphate linkages

2019 ◽  
Vol 48 (1) ◽  
pp. 63-74 ◽  
Author(s):  
Jörg Duschmalé ◽  
Henrik Frydenlund Hansen ◽  
Martina Duschmalé ◽  
Erich Koller ◽  
Nanna Albaek ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Rnase H ◽  
Modified Oligonucleotides ◽  
In Vitro Experiments ◽  
Chemical Modifications ◽  
Pharmacokinetic Properties ◽  
Target Binding ◽  
The Individual

Abstract The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3′ thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3′ and 5′ thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.

scholarly journals The structure of a class 3 nonsymbiotic plant haemoglobin fromArabidopsis thalianareveals a novel N-terminal helical extension

2014 ◽  
Vol 70 (5) ◽  
pp. 1411-1418 ◽  
Author(s):  
Brandon J. Reeder ◽  
Michael A. Hough
Keyword(s):  
Crystal Structure ◽  
Protein Sequence ◽  
Disulfide Bonds ◽  
External Environment ◽  
Tryptophan Residue ◽  
Central Cavity ◽  
Dimer Interface ◽  
Iron Coordination

Plant nonsymbiotic haemoglobins fall into three classes, each with distinct properties but all with largely unresolved physiological functions. Here, the first crystal structure of a class 3 nonsymbiotic plant haemoglobin, that fromArabidopsis thaliana, is reported to 1.77 Å resolution. The protein forms a homodimer, with each monomer containing a two-over-two α-helical domain similar to that observed in bacterial truncated haemoglobins. A novel N-terminal extension comprising two α-helices plays a major role in the dimer interface, which occupies the periphery of the dimer–dimer face, surrounding an open central cavity. The haem pocket contains a proximal histidine ligand and an open sixth iron-coordination site with potential for a ligand, in this structure hydroxide, to form hydrogen bonds to a tyrosine or a tryptophan residue. The haem pocket appears to be unusually open to the external environment, with another cavity spanning the entrance of the two haem pockets. The final 23 residues of the C-terminal domain are disordered in the structure; however, these domains in the functional dimer are adjacent and include the only two cysteine residues in the protein sequence. It is likely that these residues form disulfide bondsin vitroand it is conceivable that this C-terminal region may act in a putative complex with a partner moleculein vivo.

NAD-glycohydrolase in renal brush border membranes

1985 ◽  
Vol 249 (3) ◽  
pp. F366-F373
Author(s):  
S. A. Kempson
Keyword(s):  
Brush Border ◽  
Osmotic Shock ◽  
Phosphate Transport ◽  
Reaction Products ◽  
Brush Border Membranes ◽  
Nad Glycohydrolase ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.

scholarly journals The Metallo-β-Lactamase/β-CASP Domain of Artemis Constitutes the Catalytic Core for V(D)J Recombination

2004 ◽  
Vol 199 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Catherine Poinsignon ◽  
Despina Moshous ◽  
Isabelle Callebaut ◽  
Régina de Chasseval ◽  
Isabelle Villey ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Catalytic Domain ◽  
Single Amino Acid ◽  
In Vitro Mutagenesis ◽  
Recombination Activity ◽  
Catalytic Core ◽  
Class B ◽  
Repair Factor

The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the β-Lact homology domain, to which is appended (b) the β-CASP region, specific of members of the β-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the β-Lact and the β-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the β-Lact/β-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B β-Lact enzymes hydrolyzing β-lactam compounds.

scholarly journals Chemical modifications on siRNAs avoid Toll-like-receptor-mediated activation of the hepatic immune system in vivo and in vitro

2013 ◽  
Vol 26 (1) ◽  
pp. 35-46 ◽  
Author(s):  
R. Broering ◽  
C. I. Real ◽  
M. J. John ◽  
K. Jahn-Hofmann ◽  
L. M. Ickenstein ◽  
...  
Keyword(s):  
Immune System ◽  
Toll Like Receptor ◽  
Chemical Modifications

scholarly journals γ-glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with (glutamate-14C)glutathione

1976 ◽  
Vol 153 (2) ◽  
pp. 223-232 ◽  
Author(s):  
J S Elce ◽  
B Broxmeyer
Keyword(s):  
Rat Kidney ◽  
Rat Plasma ◽  
Transfer Reactions ◽  
Main Function ◽  
Binding Studies ◽  
Sequential Mechanism ◽  
Michaelis Constants ◽  
Solubilized Enzyme

1. The hydrolytic and transfer reactions catalysed by rat kidney-gamma-glutamyltransferase (EC 2.3.2.2) were studied in vitro with substrates [U-14C]glutamic acid-labelled glutathione and methionine. Initial-velocity patterns, isotope-exchange and binding studies were consistent with a branched non-sequential mechanism in which a γ-glutamyl-enzyme intermediate may react either with water (hydrolysis) or with methionine (γ-glutamyl transfer). 2. The Michaelis constant for glutathione in hydrolysis was 13.9 +/- 1.4 μm, for glutathione in transfer it was 113 +/- 15 μM and for methionine as substrate it was 4.7 +/- 0.7 mM. At substrate concentrations in the ranges of their respective Michaelis constants, the rate of transfer was about ten times higher than that of hydrolysis, but at concentrations of methionine approximating to the physiological (64 μM in rat plasma) the transfer is negligible. 3. The enzyme is reported to lie on the luminal surface of the proximal straight kidney tubule. In this situation, if the kinetic results obtained with the detergent-solubilized enzyme are relevant to the behavior of the enzyme in vivo, it appears likely that the main function of renal γ-glutamyltransferase is not in amino acid transport, but rather to hydrolyse glutathione in the renal filtrate.

scholarly journals Chemical Modifications of PhTX-I Myotoxin fromPorthidium hyoproraSnake Venom: Effects on Structural, Enzymatic, and Pharmacological Properties

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Salomón Huancahuire-Vega ◽  
Daniel H. A. Corrêa ◽  
Luciana M. Hollanda ◽  
Marcelo Lancellotti ◽  
Carlos H. I. Ramos ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Critical Role ◽  
High Catalytic Activity ◽  
Pharmacological Properties ◽  
Amino Acid Residues ◽  
Chemical Modifications ◽  
Specific Amino Acid ◽  
Catalytic Sites

We recently described the isolation of a basic PLA2(PhTX-I) fromPorthidium hyoprorasnake venom. This toxin exhibits high catalytic activity, inducesin vivomyotoxicity, moderates footpad edema, and causesin vitroneuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.

scholarly journals The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms.

1995 ◽  
Vol 131 (2) ◽  
pp. 371-383 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
R Gilmore
Keyword(s):  
Protein Sequence ◽  
Apoptotic Cell ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Temperature Sensitive ◽  
Microsomal Membranes ◽  
High Mannose ◽  
Eukaryotic Organisms

Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon-subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.

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