Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.     

Complexity in organic evolution

The present state of knowledge of the formation of the Compounds I of peroxidases and catalases is discussed in terms of the restrictions which must be placed upon a valid mechanism. It is likely that all Compounds I contain one oxygen atom bound to the heme-iron as in the Compound I of chloroperoxidase. Thus the formation of Compound I, obtained after molecular hydrogen peroxide and the enzyme diffuse together, involves a minimum of two bond ruptures and the formation of two new bonds. Yet this amazing reaction proceeds with an activation energy equal to or less than that for the fluidity of water. This result can only be accounted for by including at least one reversible step. Since Compound I formation requires the formation of an “inner sphere” complex, the presence or absence of water in the sixth co-ordination position of the heme-iron is of crucial importance. A comparison of the rates of ligand binding with the rate of Compound I formation indicate that the inner sphere complex leading to Compound I formation is formed by an excellent nucleophile, probably the peroxide anion, formed by a proton transfer from hydrogen peroxide. This proton cannot equilibrate with the bulk solvent. A proton derived from the active site would appear to be added to the hydroxide ion which permits a molecule of water to depart upon oxygen atom addition (or substitution) to (or at) the heme-iron. It is tentatively suggested that Compound I of catalase has a single active site per subunit molecule and that Compound I of peroxidase normally has two reactive sites.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.     

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.     

Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15 degrees C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8x10(7) M(-1) s(-1) and 1.4x10(7) M(-1) s(-1), respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2x10(5) M(-1) s(-1) and 2.7x10(6) M(-1) s(-1). Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6. 6x10(4) M(-1) s(-1) at pH 7 and 15 degrees C). Both reactions are controlled by the same ionizable group (average pK(a) of about 4.0) which has to be in its conjugated base form for reaction.     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity.

(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.     

Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Roles of water in heme peroxidase and catalase mechanisms

A water molecule is coproduced with the Compound I intermediate in the reactions of native heme peroxidases and catalases with hydrogen peroxide. As a result of water release/rebinding from/to the coproduct formation site the Compound I intermediate may exist in two forms: a "wet" form, Compound I(H(2)O), in which a water molecule is present at or near the site of coproduct water formation, and Compound I, in which the coproduct water formation site is "dry." It is postulated that the absence or presence of a water molecule at this site provides the structural basis for a redox pathway switching mechanism, such that the transition states for 2-electron equivalent reduction of Compound I intermediates are accessible in the dry form, but that in the wet form only 1-electron equivalent processes are possible, unless release of water can be stimulated. This concept provides the basis of a general mechanism in which the classical functional distinction between catalases and peroxidases, as well as the more complex behavior observed in halide oxidation and halogenation reactions, appear as particular cases in which variations in the degree of retention of water at the coproduct formation site influence Compound I reactivity.     

A kinetic study of the effects of hydrogen peroxide and pH on ascorbate oxidase.

Addition of hydrogen peroxide to ascorbate oxidase results in formation of a complex which has been analyzed kinetically. Since reduction of molecular oxygen to water by this enzyme occurs in more than one step, the peroxide complex may be a mimic for a catalytic intermediate. The properties of the complex are similar to those reported for a peroxide complex with laccase. Changes in the activity of ascorbate oxidase as a function of pH indicate the presence of a group in the enzyme with an apparent pK of 7.8 which must be protonated in order for the enzyme to function. The ascorbate K_m is known to be insensitive to pH in this region and the present work indicates the same to be true for the oxygen K_m. It would appear that the rate determining step in the catalytic mechanism involves protonation of an intermediate.     

A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.     

The catalytic mechanism of dye-decolorizing peroxidase DyP may require the swinging movement of an aspartic acid residue

The dye-decolorizing peroxidase (DyP)-type peroxidase family is a unique heme peroxidase family. The primary and tertiary structures of this family are obviously different from those of other heme peroxidases. However, the details of the structure-function relationships of this family remain poorly understood. We show four high-resolution structures of DyP (EC1.11.1.19), which is representative of this family: the native DyP (1.40 ?), the D171N mutant DyP (1.42 ?), the native DyP complexed with cyanide (1.45 ?), and the D171N mutant DyP associated with cyanide (1.40 ?). These structures contain four amino acids forming the binding pocket for hydrogen peroxide, and they are remarkably conserved in this family. Moreover, these structures show that OD2 of Asp171 accepts a proton from hydrogen peroxide in compound I formation, and that OD2 can swing to the appropriate position in response to the ligand for heme iron. On the basis of these results, we propose a swing mechanism in compound I formation. When DyP reacts with hydrogen peroxide, OD2 swings towards an optimal position to accept the proton from hydrogen peroxide bound to the heme iron.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.     

Stability testing of ligninase and Mn-peroxidase from Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium produces extracellular peroxidases (ligninase and Mn-peroxidase) believed to be involved in lignin degradation. These extracellular enzymes have also been implicated in the degradation of recalcitrant pollutants by the organism. Commercial application of ligninase has been proposed both for biomechanical pulping of wood and for wastewater treatment. In vitro stability of lignin degrading enzymes will be an important factor in determining both the economic and technical feasibility of application for industrial uses, and also will be critical in optimizing commercial production of the enzymes. The effects of a number of variables on in vitro stability of ligninase and Mn-peroxidase are presented in this paper. Thermal stability of ligninase was found to improve by increasing pH and by increasing enzyme concentration. For a fixed pH and enzyme concentration, ligninase stability was greatly enhanced in the presence of its substrate veratryl alcohol (3,4-dimethoxybenzyl alcohol). Ligninase also was found to be inactivated by hydrogen peroxide in a second-order process that is proposed to involve the formation of the unreactive peroxidase intermediate Compound III. Mn-peroxidase was less susceptible to inactivation by peroxide, which corresponds to observations by others that Compound III of Mn-peroxidase forms less readily than Compound III of ligninase.