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ABSTRACT: Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity. The selenol provides a 30-fold advantage over the thiol, but its activity is four orders of magnitude lower than that of bovine Gpx. In contrast, selenoglutaredoxin is an excellent catalyst for thiol-disulfide exchange reactions; it promotes the reduction of beta-hydroxyethyldisulfide by glutathione with a specific activity of 130 units mg(-1). This value is 1.8 times greater than that of C14S Grx1 under identical conditions, and >10(4) greater than the peroxidase activity of either enzyme. Given the facile reduction of the glutathionyl-selenoglutaredoxin adduct by glutathione, oxidation of the selenol by the alkyl hydroperoxide substrate likely limits catalytic turnover and will have to be optimized to create more effective Gpx mimics. These results highlight the challenge of generating Gpx activity in a small, generic protein scaffold, despite the presence of a well-defined glutathione binding site and the intrinsic advantage of selenium over sulfur derivatives.
ChemBioChem 07/2008; 9(10):1623-31. · 3.94 Impact Factor
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ABSTRACT: A 15-amino acid long selenopeptide (15SeP) was recently reported to possess nearly the same catalytic activity as glutathione peroxidase (Gpx) for the reduction of hydrogen peroxide by glutathione (Sun, Y., Li, T. Y., Chen, H., Zhang, K., Zheng, K. Y., Mu, Y., Yan, G. L., Li, W., Shen, J. C., and Luo, G. M. (2004) J. Biol. Chem. 279, 37235-37240). Such a finding is startling considering the high efficiency of the natural enzyme and the modest catalytic properties of most short peptides. As 15SeP had been subjected only to limited chemical characterization, we prepared it by a new route involving selenocysteine-mediated native chemical ligation. High resolution matrix-assisted laser desorption ionization mass spectrometry confirmed the identity of the reaction product, whereas circular dichroism spectroscopy showed that 15SeP assumes a random coil conformation in solution. Although low levels of peroxidase activity were detectable under standard assay conditions, the peptide is >5 orders of magnitude less active than native Gpx. Our observations are incompatible with claims ascribing remarkable catalytic properties to 15SeP and suggest that the efficiency of Gpx derives from its well defined three-dimensional structure.
Journal of Biological Chemistry 10/2007; 282(42):30518-22. · 4.77 Impact Factor
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ABSTRACT: The title compounds, 4 and 7, have been prepared from the corresponding α-amino acid derivative selenocystine (1) by the following sequence of steps: cleavage of the SeSe bond with NaBH4, p-methoxybenzyl (PMB) protection of the SeH group, Fmoc or Boc protection at the N-atom and Arndt–Eistert homologation (Schemes 1 and 2). A β3-heptapeptide 8 with an N-terminal β3-hSec(PMB) residue was synthesized on Rink amide AM resin and deprotected (‘in air’) to give the corresponding diselenide 9, which, in turn, was coupled with a β3-tetrapeptide thiol ester 10 by a seleno-ligation. The product β3-undecapeptide was identified as its diselenide and its mixed selenosulfide with thiophenol (Scheme 3). The differences between α- and β-Sec derivatives are discussed.
Helvetica Chimica Acta 08/2007; 90(9):1651 - 1666. · 1.48 Impact Factor
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ABSTRACT: Methods for the chemical synthesis of proteins have advanced considerably over the past decade. In many instances, laboratory synthesis can now be considered a viable alternative to ribosomal biosynthesis, especially when custom modifications of a protein are desired; chemical approaches guarantee virtually unlimited and tunable variation of the covalent structure of a polypeptide.
Current Opinion in Structural Biology 10/2003; 13(5):589-94. · 9.42 Impact Factor
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Angewandte Chemie International Edition 06/2003; 42(20):2275-7. · 13.45 Impact Factor
