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• W2079192580 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. 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. Glutathione peroxidase (Gpx 2The abbreviations used are: Gpx, glutathione peroxidase; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RP-HPLC, reverse-phase high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization.; EC 1.11.1.9) is a naturally occurring selenoenzyme that protects cells against oxidative damage by organic hydroperoxides (1Ursini F. Maiorino M. Brigelius-Flohé R. Aumann K.D. Roveri A. Schomburg D. Flohé L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (666) Google Scholar, 2Arthur J.R. CMLS Cell. Mol. Life Sci. 2000; 57: 1825-1835Crossref PubMed Google Scholar). The biological efficacy of the enzyme derives from catalytic efficiency approaching the diffusion limit for the reduction of hydrogen peroxide by glutathione (3Flohé L. Loschen G. Günzler W.A. Eichele E. Hoppe-Seyler's Z. Physiol. Chem. 1972; 353: 987-999Crossref PubMed Scopus (20) Google Scholar). The enzyme possesses an essential selenocysteine at its active site, and a catalytic cycle involving interconversion of the selenol (Fig. 1, ESeH), selenenic acid (ESeOH), and selenenylsulfide (ESeSR) forms of the protein has been proposed (4Ganther H.E. Kraus R.J. Methods Enzymol. 1984; 107: 593-602Crossref PubMed Scopus (86) Google Scholar). Structural (5Ren B. Huang W. Åkesson B. Ladenstein R. J. Mol. Biol. 1997; 268: 869-885Crossref PubMed Scopus (143) Google Scholar, 6Epp O. Ladenstein R. Wendel A. Eur. J. Biochem. 1983; 133: 51-69Crossref PubMed Scopus (612) Google Scholar) and functional (1Ursini F. Maiorino M. Brigelius-Flohé R. Aumann K.D. Roveri A. Schomburg D. Flohé L. Methods Enzymol. 1995; 252: 38-53Crossref PubMed Scopus (666) Google Scholar, 2Arthur J.R. CMLS Cell. Mol. Life Sci. 2000; 57: 1825-1835Crossref PubMed Google Scholar) studies suggest that the enzyme enhances the nucleophilicity of the selenol via a hydrogen bonding network with Nϵ1 of Trp148 and Nϵ2 of Gln70 and the helical dipole of the α1 helix. However, many questions remain about its detailed mechanism of action. Alternate catalytic cycles have been suggested (7Wendel, A. (1980) in Enzymatic Basis of Detoxication (Jakoby, W. B., ed) Vol. 1, pp. 333–353, Academic Press, New YorkGoogle Scholar), and the functional roles of other amino acids in the active site have yet to be elucidated. A variety of selenium-containing compounds mimic Gpx activity. For example, the enzymes subtilisin, glyceraldehyde-3-phosphate dehydrogenase, and glutathione S-transferase have been successfully converted into peroxidases by introducing selenocysteine into their active sites (8Wu Z.P. Hilvert D. J. Am. Chem. Soc. 1990; 112: 5647-5648Crossref Scopus (209) Google Scholar, 9Boschi-Muller S. Muller S. Van Dorsselaer A. Böck A. Branlant G. FEBS Lett. 1998; 439: 241-245Crossref PubMed Scopus (57) Google Scholar, 10Yu H.J. Liu J.Q. Böck A. Li J. Luo G.M. Shen J.C. J. Biol. Chem. 2005; 280: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). A protein environment is not required for antioxidant activity, however, and many low molecular weight organoselenium compounds have been shown to promote peroxide reduction (11Mugesh G. Singh H.B. Chem. Soc. Rev. 2000; 29: 347-357Crossref Scopus (596) Google Scholar, 12Mugesh G. du Mont W.W. Sies H. Chem. Rev. 2001; 101: 2125-2179Crossref PubMed Scopus (1456) Google Scholar). One such compound, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one, commonly known as ebselen, exhibits anti-inflammatory, anti-atherosclerotic, and cytoprotective properties (13Sies H. Masumoto H. Adv. Pharmacol. 1997; 38: 229-246Crossref PubMed Scopus (214) Google Scholar). These have spurred efforts to optimize ebselen and to design new Gpx mimics for potential therapeutic applications. Although significant peroxidase activity has been achieved in some cases, the efficiency of Gpx mimics generally lags far behind that of the natural enzyme. Given this, a recent study describing a 15-amino acid long selenocysteine-containing peptide with activity similar to that of native Gpx attracted our attention (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Luo and co-workers (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) prepared a series of glutathione-binding peptides by phage display and subsequently optimized their structures by computer modeling. Chemical conversion of the C-terminal serine to a selenocysteine afforded molecules that were claimed to have a homodimeric, diselenide-cross-linked, β-hairpin structure, with specific binding sites for glutathione and peroxide. More significantly, 15SeP, the most active variant, reportedly promoted the reduction of hydrogen peroxide and alkyl hydroperoxides by glutathione, with apparent second-order rate constants (kcat/Km(GSH) and kcat/Km(H2O2)) in excess of 106 m–1 s–1. The properties accorded to 15SeP are astonishing, the more so in light of the characteristic failure of small peptides to catalyze reactions with the rates and selectivities of full-length enzymes (15Matthews B.W. Craik C.S. Neurath H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4103-4105Crossref PubMed Scopus (35) Google Scholar, 16Corey M.J. Corey E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11428-11434Crossref PubMed Scopus (56) Google Scholar). Because only rudimentary chemical characterization of the selenopeptide had been reported, we resynthesized 15SeP by an unambiguous route. Upon re-examination, we found no evidence that it adopts a defined conformation in solution, nor does 15SeP possess significant Gpx activity in our hands. Rather, our results indicate that the privileged microenvironment of the Gpx active site (5Ren B. Huang W. Åkesson B. Ladenstein R. J. Mol. Biol. 1997; 268: 869-885Crossref PubMed Scopus (143) Google Scholar, 6Epp O. Ladenstein R. Wendel A. Eur. J. Biochem. 1983; 133: 51-69Crossref PubMed Scopus (612) Google Scholar) enormously enhances the intrinsic reactivity of the selenium prosthetic group. Materials—Buffers were prepared with ultrapure water. All chemicals were purchased from Sigma, Fluka, or Acros. Glutathione reductase from bakers' yeast and Gpx from bovine erythrocytes were obtained from Sigma. Peptide Synthesis—The protected peptide t-butoxycarbonyl-WPFLRHNVYGRPRA (14P) was assembled in a stepwise fashion on a 0.1-mmol scale on commercially available trityl resin preloaded with alanine using an ABI 433A peptide synthesizer (Applied Biosystems). Standard HBTU/HOBt (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N-hydroxybenzotriazole) activation protocols for Fmoc chemistry (FastMoc® protocol, Applied Biosystems) (17Knorr R. Trzeciak A. Bannwarth W. Gillessen D. Tetrahedron Lett. 1989; 30: 1927-1930Crossref Scopus (932) Google Scholar) were used. Amino acid side chains were protected as follows: Arg(Pbf) (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), Asn(trityl), His(trityl), Trp(t-butoxycarbonyl), and Tyr(t-butoxy). After the Fmoc group of the last coupled amino acid was deprotected, the peptide was capped with tert-butyl pyrocarbonate (0.4 m, 10 eq) in N-methylpyrrolidone containing N,N′-diisopropylethylamine (10 eq). Following completion of the reaction as judged by the trinitrobenzene sulfonate test (18Hancock W.S. Battersby J.E. Anal. Biochem. 1976; 71: 260-264Crossref PubMed Scopus (261) Google Scholar), the resin was washed with N-methylpyrrolidone and then with dichloromethane. The protected peptide was cleaved from the solid support by stirring in 10 ml of dichloromethane/AcOH/trifluoroethanol (3:1:1) for 2.5 h. After removal of the resin by filtration, hexane (350 ml) was added, and the solvent was evaporated. The recovered solid was resuspended in a small amount of dioxane, flash-frozen, and lyophilized. The crude protected peptide was dissolved in a 2:1 mixture of N,N′-dimethylformamide/dichloromethane (0.02 m) and converted to a thioester by adding PyBOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; 3 eq) in the presence of p-acetamidothiophenol (ArSH; 3 eq) and N,N′-diisopropylethylamine (3 eq) at 0 °C (19von Eggelkraut-Gottanka R. Klose A. Beck-Sickinger A.G. Beyermann M. Tetrahedron Lett. 2003; 44: 3551-3554Crossref Scopus (79) Google Scholar). The mixture was allowed to warm to room temperature, and the solvent was evaporated after 3 h. A small amount of water was added to the crude mixture, which was then lyophilized. The crude thioester was deprotected with 10 ml of trifluoroacetic-acid/triisopropylsilane/H2O (95:2.5:2.5) for 2.5 h at room temperature and subsequently precipitated by adding at least 8 volumes of cold diethyl ether. After centrifugation at 5000 × g for 25 min at 4 °C, the ether was decanted, and the trituration procedure was repeated twice. The crude thioester was purified by preparative reverse-phase high performance liquid chromatography (RP-HPLC) on a Macherey-Nagel C18 column (250 mm × 21 mm × 100 Å, 7 μm), eluting with a linear gradient of 5–50% solvent A (acetonitrile containing 0.05% trifluoroacetic acid) in solvent B (water containing 0.1% trifluoroacetic acid) over 110 min (flow rate of 10 ml/min). Under these conditions, the desired thioester, 14P-SAr, eluted with a retention time of ∼70 min (120 mg, corresponding to a 50% yield based on the initial resin loading): electrospray ionization mass spectrometry, [M + H]+, 1916.973 expected and 1916.972 measured; and analytical RP-HPLC (Macherey-Nagel C8 column, 250 mm × 4.6 mm × 300 Å, 5 μm; eluted with a linear gradient of 5–60% solvent A in solvent B over 45 min), Rt = 28.3 min. Selenocysteine-mediated Ligation—The thioester 14P-SAr (12 mg, 5 μmol, 1 eq) and selenocystine (1.0 mg, 3 μmol, 0.6 eq) were mixed under nitrogen in 2.5 ml of degassed 100 mm phosphate buffer (pH 7.5) containing 6 m guanidinium chloride and 5% thiophenol. The reaction was complete after 18 h as judged by liquid chromatography/mass spectrometry. Chromatography was performed with linear gradients of CH3CN and H2O containing 0.1% formic acid at a flow rate of 0.2 ml/min on a Waters Atlantis dC18-3 column (3 × 100 mm), and masses were determined at an electrospray ionization source of a Finnigan LCQ Deca ion trap mass spectrometer. After the addition of 25% AcOH (1.5 ml) and 0.1% trifluoroacetic acid (0.75 ml), the mixture was extracted four times with diethyl ether to remove excess thiophenol. The aqueous solution was centrifuged and injected onto a preparative Macherey-Nagel C8 HPLC column (250 mm × 21 mm × 300 Å, 7 μm). The selenopeptide 15SeP was eluted with a linear gradient of 5–50% solvent A in solvent B over 110 min as the diselenide dimer (6.2 mg, 25% yield; Rt =∼54 min): high resolution matrix-assisted laser desorption ionization (MALDI), [M + H]+, 3836.792 calculated and 3836.785 measured; analytical RP-HPLC (C8; 5–60% solvent A in solvent B over 45 min), Rt = 29.6 min. Circular Dichroism Spectroscopy—CD spectra were recorded using an Aviv Model 202 spectropolarimeter. Measurements were performed with 60 μm 15SeP in the presence and absence of glutathione (1 mm) in 50 mm phosphate buffer (pH 7.0) containing 100 mm NaCl, 1 mm EDTA, and 1 mm NaN3 at 25 °C. Spectra were recorded 10 times in 0.5-nm steps with a 1-s averaging time and corrected for the corresponding solvent background. Fluorescence Titration—The interaction of glutathione with 15SeP was assessed by fluorescence quenching using λex = 295 nm and λem = 345 nm as described previously (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Glutathione Peroxidase Activity—The catalytic activities of Gpx and 15SeP were determined by the method of Wendel (20Wendel A. Methods Enzymol. 1981; 77: 325-333Crossref PubMed Scopus (1238) Google Scholar). Briefly, glutathione (0.5–8 mm) was preincubated for 10 min at 37 °C with either 60 μm 15SeP or 0.6–6 nm Gpx in 50 mm phosphate buffer (pH 7.0) containing 100 mm NaCl, 1 mm EDTA, 1 mm NaN3, 250 μm NADPH, and 1 unit/ml glutathione reductase. The reaction was initiated by the addition of H2O2. Initial rates were determined by monitoring the disappearance of NADPH spectrophotometrically at 340 nm (Δϵ = 6220 m–1 cm–1) and were corrected for the background reaction measured in the absence of either 15SeP or Gpx. Luo and co-workers (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) prepared the selenopeptide 15SeP from its serine homolog via a two-step chemical modification procedure (Fig. 2a). The peptide Trp-Pro-Phe-Leu-Arg-His-Asn-Val-Tyr-Gly-Arg-Pro-Arg-Ala-Ser was first treated with phenylmethanesulfonyl fluoride to convert the C-terminal serine into a sulfonate ester. Displacement of the sulfonate with hydrogen selenide then afforded 15SeP (Trp-Pro-Phe-Leu-Arg-His-Asn-Val-Tyr-Gly-Arg-Pro-Arg-Ala-Sec). The final product, which was isolated as a diselenide dimer, was purified only by centrifugation and dialysis. Further chemical characterization was meager at best: the selenium content of 15SeP was determined by an indirect colorimetric assay, and no HPLC or mass spectroscopic data were provided to document identity or purity. We resynthesized 15SeP by a straightforward route based on selenocysteine-mediated native chemical ligation (Fig. 2b) (21Quaderer R. Sewing A. Hilvert D. Helv. Chim. Acta. 2001; 84: 1197-1206Crossref Scopus (132) Google Scholar, 22Hondal R.J. Nilsson B.L. Raines R.T. J. Am. Chem. Soc. 2001; 123: 5140-5141Crossref PubMed Scopus (220) Google Scholar, 23Gieselman M.D. Xie L. van der Donk W.A. Org. Lett. 2001; 3: 1331-1334Crossref PubMed Scopus (188) Google Scholar). The 14-amino acid long peptide Trp-Pro-Phe-Leu-Arg-His-Asn-Val-Tyr-Gly-Arg-Pro-Arg-Ala was prepared by solid-phase peptide synthesis and cleaved from the resin in protected form. Following activation of its C terminus as a thioester (19von Eggelkraut-Gottanka R. Klose A. Beck-Sickinger A.G. Beyermann M. Tetrahedron Lett. 2003; 44: 3551-3554Crossref Scopus (79) Google Scholar), side chain-protecting groups were removed, and the peptide was purified by RP-HPLC. The deprotected thioester reacted cleanly with selenocysteine under denaturing conditions in the presence of thiophenol to give 15SeP. The selenopeptide was isolated as the diselenide in 25% yield after RP-HPLC purification. As shown in Fig. 3, the synthetic material is >98% pure. High resolution MALDI mass spectrometry (Fig. 3, inset) confirmed that the product is the desired diselenide of 15SeP ([M + H]+, 3836.792 calculated and 3836.785 measured). The small contaminant visible at 29 min in the chromatogram has a mass that corresponds to the mixed diselenide between 15SeP and selenocysteine ([M + H]+, 2086.1 expected and 2086.0 measured), which should have a reactivity profile like that of the homodimer. Luo and co-workers (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) originally proposed a two-stranded, antiparallel, β-hairpin structure for 15SeP sharing “similarity with the active central structure of Gpx”, but presented no corroborating biophysical evidence. We therefore investigated the conformation of the selenopeptide by CD spectroscopy. As shown in Fig. 4, 15SeP is a random coil in 50 mm sodium phosphate buffer (pH 7), as expected for a peptide of this size. The addition of high glutathione concentrations leads to a shift in the peak minimum from 203 to ∼208 nm, suggesting either the induction of some secondary structure or aggregation. Nevertheless, attempts to quantify the interaction by fluorescence quenching afforded only small changes in the initial signal (<4%) and failed to confirm the published association constant of 3.1 × 104 m–1 (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The ability of 15SeP to promote the reduction of hydrogen peroxide by glutathione was monitored by a standard coupled assay (20Wendel A. Methods Enzymol. 1981; 77: 325-333Crossref PubMed Scopus (1238) Google Scholar). Oxidized glutathione produced in the course of the reaction was catalytically reduced in situ by NADPH and glutathione reductase. As a positive control, commercial bovine Gpx was assayed in parallel with the selenopeptide. As shown in Fig. 5a, saturation kinetics were observed when glutathione was held constant at 2.0 mm and the concentration of hydrogen peroxide was varied. For Gpx, the data afforded apparent kcat and kcat/Km(H2O2) values of 160 ± 10 s–1 and (2.4 ± 0.4) × 107 m–1 s–1, respectively. Under identical conditions, the 15SeP selenopeptide is essentially inactive, but at peptide concentrations that are 104-fold greater than those of Gpx, modest peroxidase activity was detected. The steady-state parameters for 15SeP (kcat = 0.0017 ± 0.0001 s–1 and kcat/Km(H2O2) = 11 ± 1.0 m–1 s–1) are >105 times smaller than the values we obtained for authentic Gpx and >105 times smaller than those reported by Luo and co-workers (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Consistent with a report that the Km value for glutathione is >10 mm (24Flohé L. Brand I. Biochim. Biophys. Acta. 1969; 191: 541-549Crossref PubMed Scopus (65) Google Scholar), only slight curvature was observed in analogous experiments in which the concentration of glutathione was varied and hydrogen peroxide was held constant at 200 μm (Fig. 5b). The apparent second-order rate constant kcat/Km(GSH) was therefore estimated from data obtained at low glutathione concentration. Again, however, the value for the selenopeptide (0.46 m–1 s–1) is >5 orders of magnitude smaller than the value for Gpx (9.3 × 104 m–1 s–1) and 1 million-fold smaller than that reported by Luo and co-workers (14Sun Y. Li T.Y. Chen H. Zhang K. Zheng K.Y. Mu Y. Yan G.L. Li W. Shen J.C. Luo G.M. J. Biol. Chem. 2004; 279: 37235-37240Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The basis for the enormous discrepancy between our results and the previously published data is unclear, but we are forced to conclude that selenopeptide 15SeP is an extremely poor Gpx mimic. The lack of significant 15SeP peroxidase activity is in keeping with previous findings that short peptides (<30 amino acids) typically fail to match the rates and selectivities of natural enzymes (15Matthews B.W. Craik C.S. Neurath H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4103-4105Crossref PubMed Scopus (35) Google Scholar, 16Corey M.J. Corey E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11428-11434Crossref PubMed Scopus (56) Google Scholar). There are few reliable examples of efficient peptide catalysis (25Miller S.J. Acc. Chem. Res. 2004; 37: 601-610Crossref PubMed Scopus (373) Google Scholar), and previous claims attributing extraordinary capabilities to such molecules (26Atassi M.Z. Manshouri T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8282-8286Crossref PubMed Scopus (59) Google Scholar, 27Hahn K.W. Klis W.A. Stewart J.M. Science. 1990; 248: 1544-1547Crossref PubMed Scopus (209) Google Scholar) have invariably been proven mistaken upon careful reinvestigation (16Corey M.J. Corey E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11428-11434Crossref PubMed Scopus (56) Google Scholar, 28Wells J.A. Fairbrother W.J. Otlewski J. Laskowski Jr., M. Burnier J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4110-4114Crossref PubMed Scopus (27) Google Scholar, 29Corey D.R. Phillips M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4106-4109Crossref PubMed Scopus (37) Google Scholar). Efficient catalysis generally requires accurate placement of functional groups within a precisely defined three-dimensional structure, something that is difficult to achieve with short peptide sequences. Comparison of Gpx and 15SeP indicates that the inherent reactivity of selenocysteine is enormously enhanced when the prosthetic group is placed within the active site of the enzyme. The magnitude of this activation is similar to that observed for the reactive nucleophile in the catalytic triad of serine proteases (30Carter P. Wells J.A. Nature (Lond.). 1988; 332: 564-568Crossref PubMed Scopus (532) Google Scholar)." @default.
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• W2079192580 title "Reinvestigation of a Selenopeptide with Purportedly High Glutathione Peroxidase Activity" @default.
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