FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034145974> ?p ?o ?g. }

• W2034145974 endingPage "20191" @default.
• W2034145974 startingPage "20185" @default.
• W2034145974 abstract "Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 ± 0.1) × 107m-1 s-1 (pH 7 and 15 °C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 ± 0.3) × 104m-1 s-1 and (1.7 ± 0.4) × 102m-1 s-1) and ascorbate ((1.3 ± 0.2) × 104m-1 s-1 and (8.8 ± 0.1) × 101m-1 s-1 at pH 7 and 15 °C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure. Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 ± 0.1) × 107m-1 s-1 (pH 7 and 15 °C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 ± 0.3) × 104m-1 s-1 and (1.7 ± 0.4) × 102m-1 s-1) and ascorbate ((1.3 ± 0.2) × 104m-1 s-1 and (8.8 ± 0.1) × 101m-1 s-1 at pH 7 and 15 °C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure. Catalase-peroxidases (KatGs) 1The abbreviations used are: KatGs, catalase-peroxidases; APX, ascorbate peroxidase; CCP, cytochrome c peroxidase; HRP, horseradish peroxidase; CT1 (>600 nm), long wavelength porphyrin-to-metal charge transfer band; 5-c, five-coordinate; 6-c, six-coordinate; HS, high-spin; LS, low-spin; POA, peroxoacetic acid; EPR, electron paramagnetic resonance; CD, circular dichroism; por, porphyrin. are found in archaebacteria, eubacteria, and fungi. On the basis of sequence similarities with fungal cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs), KatGs have been shown to be members of class I of the superfamily of plant, fungal, and bacterial heme peroxidases (1Welinder K.G. Curr. Opin. Struct. Biol. 1992; 2: 388-393Crossref Scopus (778) Google Scholar). Recently, the 2.0-Å; crystal structure of the KatG from the archaebacterium Haloarcula marismortui has been published (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (140) Google Scholar). It clearly showed that in KatGs the conserved proximal amino acids His, Asp, and Trp and the conserved distal amino acids Trp, Arg, and His have coordinates very similar to CCP and APX (Fig. 1). Despite this homology, class I peroxidases differ in their reactivities toward hydrogen peroxide and one-electron donors as well as in the electronic and spectral features of their redox intermediates. These differences are shown in the generalized reaction scheme of Fig. 2. In the first phase of the catalytic cycles of heme peroxidases and catalases, the ferric enzyme is oxidized by hydrogen peroxide to the redox intermediate compound I, and a water molecule is formed (see Fig. 2, Reaction 1). Compound I is the key oxidizing intermediate and is two oxidizing equivalents above that of the native ferric enzyme with an oxoferryl (FeIV=O) center in combination with either a porphyrin π-cation radical or an amino acid radical (R·+). Ascorbate peroxidases have been shown to form a π-cation radical (3Patterson W.R. Poulos T.L. Goodin D.B. Biochemistry. 1995; 34: 4342-4345Crossref PubMed Scopus (165) Google Scholar) whereas in CCP Trp-191 is the radical site (4Sivaraja M. Goodin D.B. Mauk A.G. Smith M. Hoffman B.A. Science. 1989; 245: 738-740Crossref PubMed Scopus (476) Google Scholar).Fig. 2Generalized reaction scheme of peroxidases. In the first step H2O2 is used for compound I formation (Reaction 1). Compound I is two oxidizing equivalents above that of the native enzyme with a porphyrin π-cation radical in combination with an iron(IV) center or an amino acid radical in combination with iron(IV). Compound I can react with a second H2O2 reducing the enzyme back to the ferric state (Reaction 2, catalase reaction). In the peroxidase reaction compound I is transformed in the first one-electron reduction to compound II containing either an iron(IV) center or an amino acid radical (R·) in combination with iron(III) (Reaction 3). Compound II is finally reduced back to ferric peroxidase in a second one-electron reduction (Reaction 4). Compound III (oxyperoxidase) is formed with excess H2O2. It exists as resonance structure with the iron(III)-superoxide form dominating (12Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The main difference in the enzymatic mechanism between catalases and peroxidases is compound I reduction. In a catalase cycle a second peroxide molecule is used as a reducing agent for compound I regenerating the native enzyme and releasing molecular oxygen (Reaction 2). Catalase-peroxidases have a predominant catalase activity (5Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar, 6Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1087-1096Crossref PubMed Scopus (42) Google Scholar, 7Engleder M. Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. Biochimie (Paris). 2000; 82: 211-219Crossref PubMed Scopus (20) Google Scholar, 8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 9Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar) whereas no substantial catalase activity has ever been reported for either CCP or APX. Since with hydrogen peroxide compound I reduction (k2) is much faster than compound I formation (k1), even with a stopped-flow machine it is unlikely to monitor compound I formation of wild-type KatG. Thus, under steady-state turnover conditions the ferric redox state dominates (8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar). Recently, the role of distal Trp, Arg, and His (Fig. 1) was studied in the KatGs from Escherichia coli (8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar) and from the cyanobacterium Synechocystis PCC 6803 (10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The data presented in these articles suggest that the distal His and Arg in KatGs have a role in the heterolytic cleavage of hydrogen peroxide (Reaction 1) similar to other peroxidases (12Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999Google Scholar). By contrast, the distal Trp has been shown to be essential for H2O2 oxidation, i.e. the two-electron reduction step of compound I back to the ferric protein (Reaction 2). The reasoning for this was based on the observations that in the Trp variants: (i) the catalase activity was significantly reduced (8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar) or even lost (10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), whereas (ii) the ratio of peroxidase to catalase activity was strongly increased (8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar) indicating that compound I formation was not influenced by this mutation. In the peroxidase reaction compound I is transformed in the first one-electron reduction to compound II containing either an oxoferryl (FeIV=O) center or an amino acid radical (R·+) in combination with FeIII (Fig. 2, Reaction 3). Compound II is finally reduced back to ferric peroxidase in a second one-electron reduction (Reaction 4). Both APXs and CCP cannot perform Reaction 2 and thus reduce compound I via compound II exclusively, thereby oxidizing the preferred electron donors ascorbate and cytochrome c, respectively. By contrast, in the catalase-peroxidases both the peroxidase (Reactions 1, 3, and 4) and the catalase cycle (Reactions 1 and 2) are active. The nature of KatG compound II is unclear. In earlier stopped-flow spectroscopy studies no optical evidence for a typical (red-shifted) oxoferryl-type compound II was observed when electron donors were added to compound I formed by peroxoacetic acid. The resulting intermediate exhibited similar spectral characteristics as the ferric enzyme, which was interpreted as a species containing an oxidized amino acid (R·+) in combination with FeIII (Fig. 2) (10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 13Jakopitsch C. Regelsberger G. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. J. Inorg. Biochem. 2002; 91: 78-86Crossref PubMed Scopus (23) Google Scholar). It is of note that no EPR evidence of such intermediate has been observed and that rate constants of Reaction 4 in KatGs are unknown so far. The debate about the structural requirements that enable a peroxidase to catalyze H2O2 oxidation (Reaction 2) is still going on. Here, we report on the role of a distal tyrosine, which is found in all catalase-peroxidases sequenced so far, but not in CCP and APX (Fig. 1). Exchange of Tyr-249 in Synechocystis KatG by Phe completely transformed the catalase-peroxidase to a monofunctional peroxidase, which lost its catalase activity but fully retained its peroxidase activity. As a consequence compound I formation (Fig. 1, Reaction 1) could be followed by addition of equimolar H2O2 and the calculated rate constant is shown to be similar to plant-type peroxidases. Hydrogen peroxide oxidation by Y249F (Reaction 2) was negligible, but the peroxidatic cycle was fully active. For the first time a KatG compound II with an oxoferryl-type spectrum was observed allowing the determination of rate constants of both its formation (Reaction 3) and reduction (Reaction 4). The findings are compared with similar reactions of wild-type KatG and other peroxidases and are discussed with respect to the prediction of novel covalent bonds in H. marismortui KatG (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (140) Google Scholar), which include the conserved distal tryptophan and tyrosine (Fig. 1), and the recent report about the formation of tyrosyl radical(s) in Mycobacterium tuberculosis KatG during reaction of the resting enzyme with alkyl peroxides (14Chouchane S. Girotto S. Yu S. Magliozzo R.S. J. Biol. Chem. 2002; 277: 42633-42638Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Reagents—Standard chemicals and biochemicals were obtained from Sigma Chemical Co. at the highest grade available. Expression, purification of KatGs from Synechocystis and spectrophotometric characterization of wild-type and mutant proteins were described previously (6Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1087-1096Crossref PubMed Scopus (42) Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Mutagenesis—Oligonucleotide site-directed mutagenesis was performed using PCR-mediated introduction of silent mutations as described (11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A pET-3a expression vector, that contained the cloned catalase-peroxidase gene from the cyanobacterium Synechocystis PCC 6803 (6Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1087-1096Crossref PubMed Scopus (42) Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), was used as the template for PCR. At first unique restriction sites were selected flanking the region to be mutated. The flanking primers were 5′-AAT GAT CAG GTA CCG GCC AGT AAA TG-3′ containing a KpnI restriction site and 5′-AGTGCAGACTAGTTCGGAAACG-3′ containing a SpeI restriction site. The following mutant primer with the desired mutation and a silent mutation introducing a restriction site were constructed (point mutations italicized and restriction sites underlined): 5′-TGGGATTAATTTTCGTTAATCCGGAGGGGGT GG-3′ and 5′-CACCCCCTCCGGATTAACGAAAATTAATCCCATTTG-3′ changed Tyr-249 to Phe. The fragment defined by the KpnI and SpeI restriction sites was replaced by the new construct containing the point mutation. The construct was sequenced to verify DNA changes using thermal cycle sequencing. Spectroscopic Studies—Optical spectra were recorded on a diode array spectrophotometer (Zeiss Specord S10) and a Hitachi U-3000 spectrophotometer equipped with a thermostatted cell holder. Circular dichroism studies were carried out using a JASCO J-600 spectropolarimeter. Far-UV (190-260 nm) experiments were carried out using protein concentrations of 0.15 μm, and the path length of the cuvette was 10 mm. A good signal-to-noise ratio in the CD spectra was obtained by averaging twelve scans (resolution: 1 nm; bandwidth: 1 nm; response 16 s; scan speed 20 nm/min). The protein concentration was calculated from the known amino acid composition and absorption at 280 nm according to Gill and Hippel (15Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5070) Google Scholar). Steady-state Kinetics—Catalase activity was determined polarographically in 50 mm phosphate buffer using a Clark-type electrode (YSI 5331 Oxygen Probe) inserted into a stirred water bath (YSI 5301B) at 30 °C. Alternatively, the catalase activity was measured by continuously monitoring hydrogen peroxide concentration polarographically with a platinum electrode covered with a hydrophilic membrane and fitted to the Amperometric Biosensor Detector 3001 (Universal Sensors, Inc.). The applied electrode potential at pH 7 was 650 mV, and the H2O2 electrode filling solution was prepared freshly half daily. The electrode was calibrated against known concentrations of hydrogen peroxide. All reactions were performed at 30 °C and started by the addition of KatG. One unit of catalase is defined as the amount that decomposes 1 μmol of H2O2/min at pH 7 and 30 °C. Peroxidase activity was monitored spectrophotometrically using 1 mm H2O2 (or 1 mm peroxoacetic acid) and 5 mm guaiacol (ϵ470 = 26.6 mm-1 cm-1) or 1 mmo-dianisidine (ϵ460 = 11.3 mm-1 cm-1) or 1 mm tyrosine (ϵ315 = 3.6 mm-1 cm-1) or 1 mm ascorbate (ϵ290 = 2.8 mm-1cm-1). Peroxidase activity with pyrogallol was monitored using 100 μm H2O2 and 20 mm pyrogallol (ϵ430 = 2.47 mm-1 cm-1). One unit of peroxidase is defined as the amount that decomposes 1 μmol of electron donor/min at pH 7 and 30 °C. Steady-state spectrophotometric measurements were made on a diode-array spectrophotometer Specord S10 (Zeiss) and a Hitachi Model U-3000 spectrophotometer, respectively. Transient State Kinetics—Transient state measurements were made using the model SX-18MV stopped-flow spectrophotometer from Applied Photophysics equipped with a 1-cm observation cell thermostatted at 15 °C. Calculation of pseudo first-order rate constants (kobs) from experimental traces at the Soret maximum was performed with the SpectraKinetic work station v4.38 interfaced to the instrument. The kinetics of oxidation of ferric KatG to compound I by hydrogen peroxide and peroxoacetic acid (POA) was followed in the single mixing mode at 407 nm. Ferric KatG and the peroxide were mixed to give a final concentration of 1 μm enzyme and 1-20 μm H2O2 or 5-35 μm peroxoacetic acid. The first data point was recorded 1.5 ms after mixing, and 2000 data points were accumulated. Second-order rate constants were calculated from the slope of the linear plot of pseudo first-order rate constants versus substrate concentration. Sequential-mixing stopped-flow analysis was used to measure compound I reduction by one-electron donors. In the first step the enzyme was mixed with equimolar H2O2 and, after a delay time of 100 ms where compound I was built, the intermediate was mixed with the electron donors ascorbate and tyrosine. The kinetics of compound II formation was monitored at 418 nm, the wavelength of maximum absorbance of Y249F compound II. The kinetic traces were fitted using the single exponential equation of the Applied Photophysics software and, from the slopes of the linear plots of the kobs values versus substrate concentration, the apparent second-order rate constants were obtained by linear square regression analysis. Reduction of Y249F compound II by tyrosine or ascorbate was monitored by using the following procedures. The ferric protein (4 μm) was premixed with equimolar concentration of hydrogen peroxide in 100 mm phosphate buffer (pH 7.0). After a delay time of 10 s, the formed compound II was allowed to react with varying concentrations of reducing substrates in the same buffer. The reactions were followed at 418 nm (disappearance of compound II) or at 407 nm (formation of the ferric KatG). Alternatively, 4 μm ferric KatG was premixed with equimolar concentration of hydrogen peroxide and after a delay time of 100 ms, compound I was allowed to react with varying concentrations of reducing substrates in the same buffer. The reactions were once again measured at 418 nm. The resulting biphasic curves showed the initial formation of compound II and then its subsequent reaction with ascorbate or tyrosine causing an exponential decrease in absorbance. All reactions were also investigated using the diode-array detector (Applied Photophysics PD.1) attached to the stopped-flow machine and the XScan Diode Array Scanning v1.07 software. Typically, in these experiments the KatG concentration was higher (e.g. 5 μm in the optical cell). Normal spectral data sets were also analyzed using the Pro-K simulation program from Applied Photophysics, which allows the synthesis of artificial sets of time dependent spectra as well as spectral analysis of enzyme intermediates. EPR Spectroscopy—Conventional 9-GHz EPR measurements were performed using a Bruker ER 300 spectrometer with a standard TE102 cavity equipped with a liquid helium cryostat (Oxford Instrument) and a microwave frequency counter (Hewlett Packard 5350B). Typically, the compound I samples were prepared by mixing manually 2.0 mm native enzyme (100 mm Tris/maleate buffer, pH 8.0) with an excess (10-fold) of peroxoacetic acid, directly in the 4-mm EPR tubes kept at 0 °C. The reaction was stopped by rapid immersion of the EPR tube in liquid nitrogen after 10 s. Sequence analysis of all as yet published katG genes as well as inspection of the recently published three-dimensional structure of H. marismortui KatG shows the existence of a conserved distal side tyrosine (Fig. 1). Based on the observation of continuous electron densities in the x-ray structure it was suggested that this tyrosine is covalently bonded to adjacent tryptophan and methionine (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (140) Google Scholar). In detail, bonds between Cϵ1 of Tyr-218 (Haloarcula numbering) and Cϵ2 of distal Trp-95 as well as between Cϵ2 of Tyr-218 and Sδ of Met-244 were suggested, though tryptic digestion and mass spectrometry of the reaction products were unable to prove definitely the existence of these covalent bonds in Haloarcula KatG. Neither in CCP nor in APXs this distal tyrosine is found, whereas the distal methionine is found in all KatGs and APXs but not in CCP (Fig. 1B). Fig. 3 depicts the UV-Vis and CD spectra of wild-type KatG and the variant Y249F investigated in this study. In the electronic absorption spectrum of ferric wild-type KatG, both the Soret and the CT1 band (637 nm) suggest the presence of a 5-c HS heme coexisting with a 6-c HS heme (16Heering H.A. Indiani C. Regelsberger G. Jakopitsch C. Obinger C. Smulevich G. Biochemistry. 2002; 41: 9237-9247Crossref PubMed Scopus (32) Google Scholar). In Y249F the shoulder at about 380 nm increases in intensity and the CT1 band red-shifts indicating an increase of the 5-c HS heme at the expense of 6-c HS heme. The A406/A280 ratios (i.e. Reinheitszahl) of Y249F varied between 0.49 and 0.52 compared with 0.57 and 0.61 of the wild-type protein. Thus, the Reinheitszahl of Y249F was comparable to that of other KatG variants produced so far (10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Jakopitsch C. Regelsberger G. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. J. Inorg. Biochem. 2002; 91: 78-86Crossref PubMed Scopus (23) Google Scholar). The protein yield was 50 - 80 mg of recombinant KatG from 1 liter of E. coli culture for both wild type and Y249F. The CD spectra of wild-type KatG and Y249F (Fig. 3B) are characteristic of a protein composed primarily of α-helices. Very little difference was observed between wild-type KatG and the variant. If conformational changes did occur, they must have been very localized and thus undetectable by CD experiments. Catalase and Peroxidase Activity—Recombinant wild-type KatG exhibits an overwhelming catalase activity. The polarographically measured specific catalase activity in the presence of 5 mm hydrogen peroxide is (1160 ± 55) units/mg of protein. For wild-type Synechocystis KatG a turnover number (kcat) of 3500 s-1 and a kcat/Km rate of 8.5 × 105m-1 s-1 was determined which underscores the singular status KatGs have among the heme peroxidase superfamily. However, exchange of Tyr-249 led to an almost complete loss of catalase activity. With a calculated turnover number of (6 ± 3) s-1 more than 99.8% of wild-type catalase activity was lost, and the remaining catalase activity was now similar to that determined for various horseradish peroxidase isoenzymes (2Yamada Y. Fujiwara T. Sato T. Igarashi N. Tanaka N. Nat. Struct. Biol. 2002; 9: 691-695Crossref PubMed Scopus (140) Google Scholar, 3Patterson W.R. Poulos T.L. Goodin D.B. Biochemistry. 1995; 34: 4342-4345Crossref PubMed Scopus (165) Google Scholar, 4Sivaraja M. Goodin D.B. Mauk A.G. Smith M. Hoffman B.A. Science. 1989; 245: 738-740Crossref PubMed Scopus (476) Google Scholar, 5Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar, 6Jakopitsch C. Rüker F. Regelsberger G. Dockal M. Peschek G.A. Obinger C. Biol. Chem. 1999; 380: 1087-1096Crossref PubMed Scopus (42) Google Scholar, 7Engleder M. Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. Biochimie (Paris). 2000; 82: 211-219Crossref PubMed Scopus (20) Google Scholar, 8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 9Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar, 10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999Google Scholar, 13Jakopitsch C. Regelsberger G. Furtmüller P.G. Rüker F. Peschek G.A. Obinger C. J. Inorg. Biochem. 2002; 91: 78-86Crossref PubMed Scopus (23) Google Scholar) s-1 (17Hiner A.N.P. Hernandez-Ruiz J. Garcia-Canovas F. Smith A.T. Arnao M.B. Acosta M. Eur. J. Biochem. 1995; 234: 506-512Crossref PubMed Scopus (66) Google Scholar). Most interesting was the finding that upon addition of micromolar hydrogen peroxide (1-5 μm) to 1 μm ferric Y249F KatG a red-shift in the absorbance spectrum was observed in a conventional spectrophotometer (Fig. 4). The resulting absorption bands at 418, 530, and 558 nm suggested the formation of a redox intermediate with spectral features similar to that of an oxoferryl-like compound II (12Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999Google Scholar). By contrast, in wild-type KatG the dominating redox intermediate spectrum during H2O2 degradation (10-1000 μm) has spectral features very similar to that of the ferric enzyme (8Hillar A. Peters B. Pauls R. Loboda A. Zhang H. Mauk A.G. Loewen P.C. Biochemistry. 2000; 39: 5868-5875Crossref PubMed Scopus (95) Google Scholar, 9Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar, 10Regelsberger G. Jakopitsch C. Furtmüller P.G. Rüker F. Switala J. Loewen P.C. Obinger C. Biochem. Soc. Trans. 2001; 29: 99-105Crossref PubMed Google Scholar, 11Regelsberger G. Jakopitsch C. Rüker F. Krois D. Peschek G.A. Obinger C. J. Biol. Chem. 2000; 275: 22854-22861Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Addition of higher concentrations of H2O2 (>10 μm) to ferric Y249F KatG led to the formation of an oxyferrous-like (compound III) intermediate (12Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999Google Scholar), as indicated by the appearance of absorption bands at 414, 542, and 576 nm (Fig. 4). A comparable transition of native wild-type Mycobacterium KatG to compound III has been reported (9Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar), but in these experiments very high concentrations (0.4 m) of hydrogen peroxide had to be added in order to produce a compound III spectrum with peaks at 418, 545, and 580 nm (9Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. 2000; 39: 9975-9983Crossref PubMed Scopus (101) Google Scholar). In the presence of peroxidase substrates the dominating redox intermediate had spectral features typical for an oxoferryl-type compound II (418, 530, and 558 nm). This has never been observed with both wild-type KatGs and mutants produced so far. However, the effect of mutation on the overall peroxida" @default.
• W2034145974 created "2016-06-24" @default.
• W2034145974 creator A5004506480 @default.
• W2034145974 creator A5016279871 @default.
• W2034145974 creator A5016449058 @default.
• W2034145974 creator A5044806177 @default.
• W2034145974 creator A5073002693 @default.
• W2034145974 creator A5079435712 @default.
• W2034145974 date "2003-05-01" @default.
• W2034145974 modified "2023-10-09" @default.
• W2034145974 title "Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine" @default.
• W2034145974 cites W1543953582 @default.
• W2034145974 cites W1578742674 @default.
• W2034145974 cites W1967072344 @default.
• W2034145974 cites W1977114947 @default.
• W2034145974 cites W1977602605 @default.
• W2034145974 cites W1980073565 @default.
• W2034145974 cites W1986603133 @default.
• W2034145974 cites W2000504214 @default.
• W2034145974 cites W2005986251 @default.
• W2034145974 cites W2024205751 @default.
• W2034145974 cites W2027055405 @default.
• W2034145974 cites W2028061849 @default.
• W2034145974 cites W2031209852 @default.
• W2034145974 cites W2033641503 @default.
• W2034145974 cites W2037163903 @default.
• W2034145974 cites W2040652904 @default.
• W2034145974 cites W2040883347 @default.
• W2034145974 cites W2043082155 @default.
• W2034145974 cites W2047659307 @default.
• W2034145974 cites W2048919757 @default.
• W2034145974 cites W2063473248 @default.
• W2034145974 cites W2078023361 @default.
• W2034145974 cites W2078996434 @default.
• W2034145974 cites W2085462040 @default.
• W2034145974 cites W2091091295 @default.
• W2034145974 cites W2107277439 @default.
• W2034145974 cites W2137286428 @default.
• W2034145974 cites W4232832709 @default.
• W2034145974 doi "https://doi.org/10.1074/jbc.m211625200" @default.
• W2034145974 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12649295" @default.
• W2034145974 hasPublicationYear "2003" @default.
• W2034145974 type Work @default.
• W2034145974 sameAs 2034145974 @default.
• W2034145974 citedByCount "85" @default.
• W2034145974 countsByYear W20341459742012 @default.
• W2034145974 countsByYear W20341459742013 @default.
• W2034145974 countsByYear W20341459742014 @default.
• W2034145974 countsByYear W20341459742015 @default.
• W2034145974 countsByYear W20341459742016 @default.
• W2034145974 countsByYear W20341459742017 @default.
• W2034145974 countsByYear W20341459742021 @default.
• W2034145974 countsByYear W20341459742022 @default.
• W2034145974 crossrefType "journal-article" @default.
• W2034145974 hasAuthorship W2034145974A5004506480 @default.
• W2034145974 hasAuthorship W2034145974A5016279871 @default.
• W2034145974 hasAuthorship W2034145974A5016449058 @default.
• W2034145974 hasAuthorship W2034145974A5044806177 @default.
• W2034145974 hasAuthorship W2034145974A5073002693 @default.
• W2034145974 hasAuthorship W2034145974A5079435712 @default.
• W2034145974 hasBestOaLocation W20341459741 @default.
• W2034145974 hasConcept C153911025 @default.
• W2034145974 hasConcept C161790260 @default.
• W2034145974 hasConcept C162008176 @default.
• W2034145974 hasConcept C181199279 @default.
• W2034145974 hasConcept C185592680 @default.
• W2034145974 hasConcept C2776165026 @default.
• W2034145974 hasConcept C2778979269 @default.
• W2034145974 hasConcept C2780443040 @default.
• W2034145974 hasConcept C55493867 @default.
• W2034145974 hasConcept C58431802 @default.
• W2034145974 hasConcept C86803240 @default.
• W2034145974 hasConceptScore W2034145974C153911025 @default.
• W2034145974 hasConceptScore W2034145974C161790260 @default.
• W2034145974 hasConceptScore W2034145974C162008176 @default.
• W2034145974 hasConceptScore W2034145974C181199279 @default.
• W2034145974 hasConceptScore W2034145974C185592680 @default.
• W2034145974 hasConceptScore W2034145974C2776165026 @default.
• W2034145974 hasConceptScore W2034145974C2778979269 @default.
• W2034145974 hasConceptScore W2034145974C2780443040 @default.
• W2034145974 hasConceptScore W2034145974C55493867 @default.
• W2034145974 hasConceptScore W2034145974C58431802 @default.
• W2034145974 hasConceptScore W2034145974C86803240 @default.
• W2034145974 hasIssue "22" @default.
• W2034145974 hasLocation W20341459741 @default.
• W2034145974 hasOpenAccess W2034145974 @default.
• W2034145974 hasPrimaryLocation W20341459741 @default.
• W2034145974 hasRelatedWork W1979238481 @default.
• W2034145974 hasRelatedWork W1982065950 @default.
• W2034145974 hasRelatedWork W1987286916 @default.
• W2034145974 hasRelatedWork W2008982968 @default.
• W2034145974 hasRelatedWork W2058400169 @default.
• W2034145974 hasRelatedWork W2079514196 @default.
• W2034145974 hasRelatedWork W2381206075 @default.
• W2034145974 hasRelatedWork W4320059643 @default.
• W2034145974 hasRelatedWork W2107972437 @default.
• W2034145974 hasRelatedWork W2523806502 @default.
• W2034145974 hasVolume "278" @default.

• next