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• W2018676388 abstract "The reactions of the fungal enzymesArthromyces ramosus peroxidase (ARP) andPhanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H2O2) have been studied. Both enzymes exhibited catalase activity with hyperbolic H2O2 concentration dependence (Km ≈ 8–10 mm,kcat ≈ 1–3 s−1). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, ki ≈ 19 × 10−3 s−1; ARP, ki ≈ 1.6 × 10−3 s−1. Compound III (oxyperoxidase) was detected as the majority species after the addition of H2O2 to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H2O2. A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I·H2O2] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H2O2, and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily. The reactions of the fungal enzymesArthromyces ramosus peroxidase (ARP) andPhanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H2O2) have been studied. Both enzymes exhibited catalase activity with hyperbolic H2O2 concentration dependence (Km ≈ 8–10 mm,kcat ≈ 1–3 s−1). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, ki ≈ 19 × 10−3 s−1; ARP, ki ≈ 1.6 × 10−3 s−1. Compound III (oxyperoxidase) was detected as the majority species after the addition of H2O2 to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H2O2. A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I·H2O2] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H2O2, and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily. hydrogen peroxide initial enzyme activity residual enzyme activity enzyme remaining at end of reaction 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) ascorbate peroxidase A. ramosus peroxidase horseradish peroxidase lignin peroxidase superoxide radical concentration of oxygen produced partition ratio tetranitromethane initial rate of H2O2decomposing activity Peroxidases (donor: hydrogen peroxide oxidoreductases) are ubiquitous enzymes that catalyze the oxidation of substrate at the expense of hydrogen peroxide (H2O2).1The heme peroxidases have been classified into two distinct groups, termed the animal (found only in animals) and plant (found in plants, fungi, and prokaryotes) superfamilies (1Dunford H.B. Heme Peroxidases. Wiley-VCH, New York1999: 281-308Google Scholar). The plant peroxidases, which share similar overall protein folds and specific features, such as catalytically essential histidine and arginine residues in their active sites, have been subdivided into three classes on the basis of sequence comparison (2Welinder K.G. Eur. J. Biochem. 1985; 151: 497-504Crossref PubMed Scopus (167) Google Scholar, 3Welinder K.G. Curr. Opin. Struct. Biol. 1992; 2: 388-393Crossref Scopus (778) Google Scholar). In class I are intracellular enzymes including yeast cytochrome c peroxidase, ascorbate peroxidase (APX) from plants, and bacterial gene-duplicated catalase-peroxidases (4Welinder K.G. Biochim. Biophys. Acta. 1991; 1080: 215-220Crossref PubMed Scopus (151) Google Scholar). Class III contains the secretory plant peroxidases such as those from horseradish (HRP), barley, or soybean. These peroxidases seem to be biosynthetic enzymes involved in processes such as plant cell wall formation and lignification. Class II consists of the secretory fungal peroxidases such as lignin peroxidase (LiP) from Phanerochaete chrysosporium, manganese peroxidase from the same source, andCoprinus cinereus peroxidase or Arthromyces ramosus peroxidase (ARP), which have been shown to be essentially identical in both sequence and properties (5Kjalke M. Andersen M.B. Schneider P. Christensen B. Schülein M. Welinder K.G. Biochim. Biophys. Acta. 1992; 1120: 248-256Crossref PubMed Scopus (91) Google Scholar). The main role of class II peroxidases appears to be the degradation of lignin in wood. All peroxidases studied so far share much the same catalytic cycle that proceeds in three distinct and essentially irreversible steps (6Dunford H.B. Everse J. Everse K.E. Grisham M.B. Peroxidases in Chemistry and Biology. CRC Press, Boca Raton, FL1990: 1-24Google Scholar) and is often referred to as the “peroxidase ping-pong.” The resting ferric enzyme reacts with H2O2 in a two-electron process to generate the intermediate known as compound I. Compound I is discharged in two sequential single-electron reactions with reducing substrate yielding radical products, which are often highly reactive, and water. The first reduction step results in the formation of another enzyme intermediate, compound II. In the final step compound II is reduced back to ferric peroxidase. The peroxidase ping-pong provides an adequate description of the peroxidase reaction; however, continuing work has revealed some limitations of the basic model. The compound I reduction steps have been shown to consist of reversible substrate binding followed by substrate oxidation (7Rodrı́guez-López J.N. Gilabert M.A. Tudela J. Thorneley R.N.F. Garcı́a-Cánovas F. Biochemistry. 2000; 39: 13201-13209Crossref PubMed Scopus (125) Google Scholar). The formation and nature of compound I have been studied intensively. Both a neutral peroxidase-peroxide complex and a charged complex (known as compound 0) have been observed (8Rodrı́guez-López J.N. Lowe D.J. Hernández-Ruiz J. Hiner A.N.P. Garcı́a-Cánovas F. Thorneley R.N.F. J. Am. Chem. Soc. 2001; 123: 11838-11847Crossref PubMed Scopus (280) Google Scholar), and variations have been identified in the electronic structures of the compound Is of different peroxidases (9Dolphin D. Forman A. Borg D.C. Fajer J. Felton R.H. Rec. Res. Dev. Agric. Food Chem. 1971; 68: 614-618Google Scholar, 10Yonetani T. Schleyer H. Ehrenberg A. J. Biol. Chem. 1966; 241: 3240-3243Abstract Full Text PDF PubMed Google Scholar, 11Sivaraja M. Goodin D.B. Smith M. Hoffman B.M. Science. 1989; 245: 738-740Crossref PubMed Scopus (476) Google Scholar, 12Hiner A.N.P. Martı́nez J.I. Arnao M.B. Acosta M. Turner D.D. Raven E.L. Rodrı́guez-López J.N. Eur. J. Biochem. 2001; 268: 3091-3098Crossref PubMed Scopus (62) Google Scholar, 13Blodig W. Doyle W.A. Smith A.T. Winterhalter K. Choinowski T. Piontek K. Biochemistry. 1998; 37: 8832-8838Crossref PubMed Scopus (76) Google Scholar, 14Converso D.A. Fernández M.E. Arch. Biochem. Biophys. 1998; 357: 22-26Crossref PubMed Scopus (6) Google Scholar). Furthermore, certain peroxidases have been found to utilize H2O2 to reduce compound I when no other substrate is available. Foremost among the enzymes capable of this are the catalase-peroxidases, which act as highly efficient catalases (kcat/Km ≈ 106m−1 s−1) (15Claiborne A. Fridovich I. J. Biol. Chem. 1979; 254: 4245-4252Abstract Full Text PDF PubMed Google Scholar,16Obinger C. Regelsberger G. Strasser G. Burner U. Peschek G.A. Biochem. Biophys. Res. Commun. 1997; 235: 545-552Crossref PubMed Scopus (59) Google Scholar). Unexpectedly, APX appears incapable of turnover in the presence of only H2O2 (17Hiner A.N.P. Rodriguez-Lopez J.N. Arnao M.B. Lloyd Raven E. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2000; 348: 321-328Crossref PubMed Scopus (85) Google Scholar) despite also being classified in class I. HRP, from class III, has been shown to possess catalase activity, albeit with much lower efficiency (kcat/Km ≈ 102–103m−1s−1) than the catalase-peroxidases (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar), but to our knowledge, no data are available on catalase activity in class II peroxidases. HRP compound I can also carry out a single-electron reduction of H2O2 to generate compound II and superoxide radical (O2⨪). Reaction of compound II with more H2O2 yields compound III (a complex between ferric peroxidase and O2⨪, also known as oxyperoxidase). Catalase activity and compound III formation result in enzyme turnover in the absence of normal reducing substrates; however, a further reaction with H2O2 has been identified which leads to progressive irreversible enzyme inactivation. In the case of HRP, the complex between compound I and peroxide ([compound I·H2O2]) has been identified unambiguously as the pivotal point connecting the three simultaneous pathways (19Arnao M.B. Acosta M. del Rio J.A. Varón R. Garcı́a-Cánovas F. Biochim. Biophys. Acta. 1990; 1041: 43-47Crossref PubMed Scopus (261) Google Scholar,20Rodriguez-Lopez J.N. Hernández-Ruiz J. Garcı́a-Cánovas F. Thorneley R.N.F. Acosta M. Arnao M.B. J. Biol. Chem. 1997; 272: 5469-5476Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Thus, the peroxidase ping-pong mechanism can be replaced by an alternative catalytic cycle (Scheme FSI) when no substrate other than H2O2 is present. Studies on the susceptibility of different peroxidases to H2O2 suggest that all will be sensitive to inactivation to a greater or lesser extent. Even catalase-peroxidase loses activity after repeated exposure to H2O2(21Obinger C. Regelsberger G. Pircher A. Sevcik-Klöckler A. Strasser G. Peschek G.A. Peschek G.A. The Phototrophic Prokaryotes. Kluwer Academic Publishers, New York1999: 719-731Crossref Google Scholar) with inactivation suggested to occur at the level of the [compound I·H2O2] complex. Previous studies on LiP compound III found comparatively facile degradation of the heme by H2O2 (which resulted in loss of activity), but aspects of this process proved rather contentious (22Wariishi H. Gold M.H. FEBS Lett. 1989; 243: 165-168Crossref Scopus (90) Google Scholar, 23Cai D. Tien M. Biochem. Biophys. Res. Commun. 1989; 162: 464-469Crossref PubMed Scopus (39) Google Scholar, 24Wariishi H. Gold M.H. J. Biol. Chem. 1990; 265: 2070-2077Abstract Full Text PDF PubMed Google Scholar, 25Wariishi H. Marquez L.A. Dunford H.B. Gold M.H. J. Biol. Chem. 1990; 265: 11137-11142Abstract Full Text PDF PubMed Google Scholar, 26Cai D. Tien M. J. Biol. Chem. 1992; 267: 11149-11155Abstract Full Text PDF PubMed Google Scholar, 27Cai D. Tien M. Biochemistry. 1990; 29: 2085-2091Crossref PubMed Scopus (17) Google Scholar). A reaction model was developed which suggested that the inactivation of LiP might occur from compound III or a modified compound III species (24Wariishi H. Gold M.H. J. Biol. Chem. 1990; 265: 2070-2077Abstract Full Text PDF PubMed Google Scholar). Following on from our work with class I (12Hiner A.N.P. Martı́nez J.I. Arnao M.B. Acosta M. Turner D.D. Raven E.L. Rodrı́guez-López J.N. Eur. J. Biochem. 2001; 268: 3091-3098Crossref PubMed Scopus (62) Google Scholar, 17Hiner A.N.P. Rodriguez-Lopez J.N. Arnao M.B. Lloyd Raven E. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2000; 348: 321-328Crossref PubMed Scopus (85) Google Scholar) and class III peroxidases (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar, 19Arnao M.B. Acosta M. del Rio J.A. Varón R. Garcı́a-Cánovas F. Biochim. Biophys. Acta. 1990; 1041: 43-47Crossref PubMed Scopus (261) Google Scholar, 20Rodriguez-Lopez J.N. Hernández-Ruiz J. Garcı́a-Cánovas F. Thorneley R.N.F. Acosta M. Arnao M.B. J. Biol. Chem. 1997; 272: 5469-5476Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Arnao M.B. Acosta M. del Rio J.A. Garcı́a-Cánovas F. Biochim. Biophys. Acta. 1990; 1038: 85-89Crossref PubMed Scopus (169) Google Scholar, 29Acosta M. Arnao M.B. Hernández-Ruiz J. Garcı́a-Cánovas F. Welinder K.G. Rasmussen S.K. Penel C. Greppin H. Plant Peroxidases: Biochemistry and Physiology. University of Geneva, Geneva1993: 201-205Google Scholar, 30Acosta M. Arnao M.B. del Rio J.A. Garcı́a-Cánovas F. Lobarzewski H. Greppin H. Penel C. Gaspar T. Biochemical, Molecular and Physiological Aspects of Plant Peroxidases. University of Geneva, Geneva1991: 175-184Google Scholar, 31Acosta M. Hernández-Ruiz J. Garcı́a-Cánovas F. Rodrı́guez-López J.N. Arnao M.B. Obinger C. Burner U. Ebermann R. Penel C. Greppin H. Plant Peroxidases: Biochemistry and Physiology. University of Geneva, Geneva1996: 76-81Google Scholar, 32Hiner A.N.P. Hernández-Ruiz J. Garcı́a-Cánovas F. Smith A.T. Arnao M.B. Acosta M. Eur. J. Biochem. 1995; 234: 506-512Crossref PubMed Scopus (67) Google Scholar, 33Hiner A.N.P. Hernández-Ruiz J. Arnao M.B. Garcı́a-Cánovas F. Acosta M. Biotechnol. Bioeng. 1996; 50: 655-662Crossref PubMed Scopus (94) Google Scholar, 34Arnao M.B. Hernández-Ruiz J. Varón R. Garcı́a-Cánovas F. Acosta M. J. Mol. Catal. A Chem. 1995; 104: 179-191Crossref Scopus (12) Google Scholar, 35Hiner A.N.P. Hernández-Ruiz J. Rodrı́guez-López J.N. Arnao M.B. Varón R. Garcı́a-Cánovas F. Acosta M. J. Biol. Inorg. Chem. 2001; 6: 504-516Crossref PubMed Scopus (47) Google Scholar, 36Arnao M.B. Garcı́a-Cánovas F. Acosta M. Biochem. Mol. Biol. Internat. 1996; 39: 97-107PubMed Google Scholar, 37Hernández-Ruiz J. Rodrı́guez-López J.N. Garcı́a-Cánovas F. Acosta M. Arnao M.B. Biochim. Biophys. Acta. 2000; 1478: 78-88Crossref PubMed Scopus (9) Google Scholar, 38Hiner A.N.P. Hernández-Ruiz J. Williams G.A. Arnao M.B. Garcı́a-Cánovas F. Acosta M. Arch. Biochem. Biophys. 2001; 392: 295-302Crossref PubMed Scopus (54) Google Scholar), we have now examined the class II enzymes, LiP and ARP. Apart from observing catalase activity and compound III formation and inactivation in fungal peroxidases, another important aim of this work was to ascertain whether a model for turnover in the presence of only H2O2 which essentially unifies the behavior of all or most peroxidases may likely exist, just as the peroxidase ping-pong describes activity with reducing substrates even though these substrates can be so varied. We have, therefore, systematically examined the enzymes using a set of experimental procedures that have proved very successful for the determination of the controlling kinetic parameters under such conditions. Further impetus for the present study was provided by the important physiological implications of the reactions of LiP under highly oxidizing conditions and because ARP is a potentially important commercial alternative to HRP. Directed evolution techniques have previously been applied to ARP (39Cherry J.R. Lamsa M.H. Schneider P. Vind J. Svendsen A. Jones A. Pedersen A.H. Nat. Biotechnol. 1999; 17: 379-384Crossref PubMed Scopus (260) Google Scholar) to produce an enzyme that is hyperresistant to conditions of high pH, temperature, and H2O2 concentrations. A better understanding of H2O2-dependent turnover and inactivation may point to new strategies to further enhance resistance during applications. Nonglycosylated recombinant LiP isoenzyme H8 (LiP) from P. chrysosporium was expressed in Escherichia coli, and the polypeptide was recovered from inclusion bodies and refolded in vitro in the presence of heme, calcium ions, and glutathione (40Doyle W.A. Smith A.T. Biochem. J. 1996; 315: 15-19Crossref PubMed Scopus (92) Google Scholar). After purification the preparation had an RZ (A409 nm/A280 nm) = 1.8 and was homogeneous by SDS-PAGE. Recombinant LiP so far has exhibited behavior identical to that of the natural fungal enzyme (40Doyle W.A. Smith A.T. Biochem. J. 1996; 315: 15-19Crossref PubMed Scopus (92) Google Scholar,41Doyle W.A. Blodig W. Veitch N.C. Piontek K. Smith A.T. Biochemistry. 1998; 37: 15097-15105Crossref PubMed Scopus (213) Google Scholar). ARP (lot 48H0555, RZ (A405 nm/A275 nm) = 2.5) and HRP (type IX, RZ (A403 nm/A275 nm) = 3.2) were obtained from Sigma as lyophilized powders. The HRP preparation has been characterized previously (33Hiner A.N.P. Hernández-Ruiz J. Arnao M.B. Garcı́a-Cánovas F. Acosta M. Biotechnol. Bioeng. 1996; 50: 655-662Crossref PubMed Scopus (94) Google Scholar) by isoelectric focusing as a single band with pI 8.5 and confirmed to be isoenzyme C. ARP was characterized similarly as a single peroxidase band with pI 3.5. Enzyme concentrations were determined spectrophotometrically using ε403 nm = 100 mm−1cm−1 (for HRP-C), ε409 nm = 168 mm−1 cm−1 (for LiP), and ε405 nm = 109 mm−1cm−1 (for ARP). Bovine erythrocyte superoxide dismutase (product code S-2515) was purchased from Sigma as a lyophilized powder containing 4,200 units mg−1. H2O2 (30% by volume) and buffer substances (analytical reagent grade) were obtained from Merck. The concentration of H2O2 was determined using ε240 nm = 43.6 m−1cm−1. ABTS in the form of the crystallized diammonium salt was supplied by Roche Molecular Biochemicals, and its concentration was measured spectrophotometrically using ε340 nm = 36 mm−1 cm−1. Tetranitromethane (TNM) and manganese (II) chloride were from Aldrich. All solutions were prepared using deionized water drawn from a Milli-Q system (Millipore). Oxygen production was measured using a Clark-type electrode coupled to a Hansatech (Kings Lynn, Cambs., UK) CB1D oxygraph unit. The equipment was calibrated using the tyrosinase/4-tert-butylcatechol method (42Rodrı́guez-López J.N. Ros-Martı́nez J.R. Varón R. Garcı́a-Cánovas F. Anal. Biochem. 1992; 202: 356-360Crossref PubMed Scopus (71) Google Scholar). The temperature of the reaction chamber was controlled at 25 ± 0.1 °C using a Haake circulating water bath. Nitrogen was bubbled through the reaction medium to remove dissolved oxygen. A base-line rise in oxygen concentration of less than 0.5 μm min−1without enzyme was obtained. The reaction medium (2 ml, total volume) contained H2O2 at the appropriate concentrations (see “Results”) in the following buffers: 50 mm sodium acetate (pH 3.0), 50 mm sodium citrate, (pH 4.5 and 5.5), and 50 mm sodium phosphate (pH 6.5, 7.0, and 7.5). The reactions were started by the addition of peroxidase. Additional reagents (Mn2+, TNM, and superoxide dismutase) were added as required. Two types of experiment were performed using the system described. Values ofV0 were determined at short reaction times in the initial linear phase of oxygen production. The initial linear phase lasted ∼10–30 s; departure from linearity at later times indicated that enzyme inactivation was being observed, and such data were therefore not used in calculations. Values ofVmax and Km were obtained by nonlinear regression of a hyperbolic function to a plot ofV0 against [H2O2] using the program SigmaPlot for Windows (version 2, Jandel Scientific Software, San Rafael, CA). These data could be fitted using the Michaelis-Menten equation, which in this case takes the following form (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar),d[O2]dt=k3[E]T[H2O2]K2+[H2O2]Equation 1 where Km = K2 andVmax =k3[E]T (thusk3 = kcat), the steps to which k3 and K2 refer being shown in Scheme FSI. The reaction end point was reached when no further O2 production was observed. In all experiments, the final concentration of oxygen produced ([O2]∞) was much less than 0.24 mm (i.e. the oxygen concentration in air-saturated medium at 25 °C). Peroxidase was inactivated at 25 °C in 100-μl incubations in buffers similar to those used in the oxygraph. Each incubation contained 1 μm enzyme and H2O2 at different concentrations (giving the desired [H2O2] to [peroxidase] ratios). When the reaction was complete, the peroxidase activity was measured spectrophotometrically by the increase in the absorbance at 414 nm (ε = 31.1 mm−1 cm−1) in an assay system comprising 10 mm ABTS and 5 mmH2O2 in 50 mm sodium phosphate buffer (pH 6.5). The residual enzyme activity (AR) (expressed as percent) was taken as the activity remaining at the end of the reaction (At) compared with the initial activity (A0). Assays were recorded on a PerkinElmer Life Sciences Lambda-2S UV-visible spectrophotometer. The temperature was controlled at 25 ± 0.1 °C using a Haake circulating water bath. The partition ratio (r, the number of turnovers with H2O2 before the enzyme was inactivated;r = (k3 +k4)/ki ≈k3/ki , becausek3 ≫ k4 (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar)) was calculated by inserting the value of [H2O2]/[peroxidase] at which %AR = 0 in graphical representations of %AR against [H2O2]/[peroxidase] into Equation 2(18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar). AR=1−12r+2[H2O2][E]0Equation 2 The inactivation of ARP was followed against time at pH 7.5. 1 μm ARP was incubated with 0, 1, 2, 5, 10, 20, and 50 mmH2O2. At appropriate times after addition of the enzyme, aliquots were removed, and the activity with ABTS was determined. The conditions used were such that the [H2O2] added with the enzyme aliquot, which varied with time as inactivation proceeded, did not affect the assayed activity. The AR (%) was calculated for each time point and H2O2 concentration. The data were plotted and fits to a first-order exponential decay to obtain values of kobs (observed rate constants of inactivation) were made using SigmaPlot. The rate constant of inactivation (ki ) and dissociation constant (KI ) were calculated from a hyperbolic secondary plot of kobs against [H2O2] using the Michaelis-Menten equation in the form (17Hiner A.N.P. Rodriguez-Lopez J.N. Arnao M.B. Lloyd Raven E. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2000; 348: 321-328Crossref PubMed Scopus (85) Google Scholar) shown in Equation 3. kobs=ki[H2O2]KI+[H2O2]Equation 3 The UV-visible spectral changes that occurred over time to peroxidase (LiP or ARP) upon addition of H2O2 (100 or 500 equivalents, respectively) were observed at pH 3.0 and pH 7.0 using a PerkinElmer Life Sciences Lambda-2S spectrophotometer. The reaction of 0.8 μm LiP with 5 μm H2O2 was measured at pH 4.0 and pH 6.0 in stopped-flow experiments done on an Applied Photophysics (Leatherhead, Surrey, UK) SX18.MV stopped-flow instrument fitted with a PDA.1 photodiode array detector. The temperature was controlled at 25 ± 0.1 °C using a Neslab circulating water bath. The multivariate data sets from the stopped-flow were fitted globally using the program Pro/K (Applied Photophysics). Both LiP and ARP showed clear catalase activity. The H2O2 concentration dependence of the initial rates (V0) of O2 production by the two fungal enzymes, shown in Fig.1, demonstrated that both exhibited saturation kinetics. From the curves, values ofK2 (Km ) andk3 (kcat) (see Scheme FSI) could be obtained using Equation 1. Fig. 1A shows the saturation curve for LiP with K2 = 8.6 ± 0.4 mm and k3 = 2.87 ± 0.21 s−1; in B the corresponding values for ARP wereK2 = 10.2 ± 2.3 mm andk3 = 1.15 ± 0.09 s−1. Thus both LiP and ARP had kinetic parameters for O2 generation of an order of magnitude similar to those determined previously for HRP-C (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar) and HRP-A2 (35Hiner A.N.P. Hernández-Ruiz J. Rodrı́guez-López J.N. Arnao M.B. Varón R. Garcı́a-Cánovas F. Acosta M. J. Biol. Inorg. Chem. 2001; 6: 504-516Crossref PubMed Scopus (47) Google Scholar). However, during experiments it was noticed that the values of V0 for LiP were maintained for only a very short time and that O2 production essentially ceased in ∼10 min, whereas for ARP it took about 1 h for activity to be lost. This suggested that LiP was being inactivated more rapidly than ARP. Determinations of the total O2 gas produced by a given amount of enzyme before its inactivation (i.e. the infinite product of the catalase reaction) confirmed that LiP lost activity much more rapidly than ARP. The values of the partition ratios (r), obtained from the slopes of the straight line fits to plots of [O2]∞ against [peroxidase] (not shown) (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar) indicated that LiP (r = 155 ± 25) was more sensitive to inactivation by H2O2 than ARP (r = 620 ± 60). Additionally, using the relationship r ≈k3/ki (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar) (see SchemeFSI), it was calculated that the inactivation constant of LiP (ki = (18.5 ± 2.3) × 10−3 s−1) was 10-fold higher than for ARP (ki = (1.85 ± 0.18) × 10−3 s−1). Thus the ki of ARP was approximately equivalent to the value for HRP-C (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar), but LiP was inactivated much more rapidly (TableI).Table IRate constants for the catalase-like activity and inactivation of LiP and ARP compared with those of HRP-CEnzymeConstants from oxygen measurementsConstants from inactivation studiesK21-aSee Equation 1.k31-aSee Equation 1.r1-bThe partition ratio,r = (k3 +k4)/ki wherek3 ≫ k4, 4 ≈k3/ki.kiki1-cSee Equation 3.KI1-cSee Equation 3.r1-bThe partition ratio,r = (k3 +k4)/ki wherek3 ≫ k4, 4 ≈k3/ki.mms−1×10−3s−1×10−3 s−1mmLiP8.6 ± 0.42.87 ± 0.21155 ± 2518.5 ± 2.320.0 ± 3.06.1 ± 0.4565 ± 15ARP10.2 ± 2.31.15 ± 0.09620 ± 601.85 ± 0.181.37 ± 0.273.83 ± 0.35425 ± 35HRP-C4.0 ± 0.61-dFrom Ref. 18.1.78 ± 0.121-dFrom Ref. 18.700 ± 1001-dFrom Ref. 18.2.5 ± 0.21-dFrom Ref. 18.3.92 ± 0.061-eFrom Ref. 19.1.3 ± 0.21-fFrom Ref. 32.624 ± 401-fFrom Ref. 32.1-a See Equation 1.1-b The partition ratio,r = (k3 +k4)/ki wherek3 ≫ k4, 4 ≈k3/ki.1-c See Equation 3.1-d From Ref. 18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar.1-e From Ref. 19Arnao M.B. Acosta M. del Rio J.A. Varón R. Garcı́a-Cánovas F. Biochim. Biophys. Acta. 1990; 1041: 43-47Crossref PubMed Scopus (261) Google Scholar.1-f From Ref. 32Hiner A.N.P. Hernández-Ruiz J. Garcı́a-Cánovas F. Smith A.T. Arnao M.B. Acosta M. Eur. J. Biochem. 1995; 234: 506-512Crossref PubMed Scopus (67) Google Scholar. Open table in a new tab Both ARP and LiP were more effective catalases at neutral than at acidic pH (data not shown). The apparent pKa ≈ 6 observed for ARP was quite similar to the value with HRP-C (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar), suggesting the involvement of the conserved distal histidine residue and that catalase activity is an enzymatic reaction. It has been shown recently that O2⨪ scavengers (superoxide dismutase, TNM, and Mn2+) have only limited effects on HRP-C catalase activity (38Hiner A.N.P. Hernández-Ruiz J. Williams G.A. Arnao M.B. Garcı́a-Cánovas F. Acosta M. Arch. Biochem. Biophys. 2001; 392: 295-302Crossref PubMed Scopus (54) Google Scholar, 43Baker C.J. Deahl K. Domek J. Orlandi E.W. Arch. Biochem. Biophys. 2000; 382: 232-237Crossref PubMed Scopus (34) Google Scholar). Fig.2 shows the effects of O2⨪scavengers on ARP. Superoxide dismutase and TNM, which generate O2 and H2O2 from O2⨪, slightly increased O2 production. In contrast Mn2+, which regenerates H2O2 but does not yield O2, slightly reduced the level of O2 released. Thus it appears that ARP, like HRP-C, produces O2 in an enzymatic reaction and not, except for a very minor component, as the result of the chemical disproportionation of O2⨪ in solution. Incubation experiments over a range of H2O2 concentrations and pH values were used to probe further the inactivation of LiP and ARP. In Fig.3 it can be seen that LiP (A) was more sensitive to H2O2 than was ARP (Fig.3B), as expected from the O2 measurements above. The insets show that both enzymes were more sensitive at acidic than at neutral pH. The values of r obtained here, calculated using Equation 2, were in reasonable agreement with those from oxygen measurements at the same pH (Table I). This suggests that, as for HRP-C (18Hernández-Ruiz J. Arnao M.B. Hiner A.N.P. Garcı́a-Cánovas F. Acosta M. Biochem. J. 2001; 354: 107-114Crossref PubMed Scopus (162) Google Scholar), the level of turnover through the catalase reaction is correlated strongly with protection of the peroxidase against inactivation by H2O2. The kinetics of inactivation were measured directly by following the time-dependent fall in peroxidase activity, determined using ABTS, as a function of H2O2concentration. In Fig. 4A the inactivation curves for ARP are shown, and in B the H2O2 concentration-dependent saturation curve from which values of ki = (1.37 ± 0.27) × 10−3 s−1 andKI = 3.83 ± 0.35 mm were calculated (see Equation 3). The equivalent experiments performed for LiP (data not shown) yielded values of ki = (20.0 ± 3.0) × 10−3 s−1 andKI = 6.1 ± 0.45 mm. In both cases the values of ki were in good agreement with those calculated independently from the oxygraph data. The value of KI is related to theK2 obtained from oxygen measurements. However, the longer periods needed" @default.
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• W2018676388 title "Reactions of the Class II Peroxidases, Lignin Peroxidase andArthromyces ramosus Peroxidase, with Hydrogen Peroxide" @default.
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