Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis.

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.

Mechanisms for protection against inactivation of manganese peroxidase by hydrogen peroxide.

It has been reported that cation radicals of aromatic substrates maintain the active form of lignin peroxidase by oxidatively converting compound III, generated during peroxidase turnover, into ferric enzyme (D. P. Barr and S. D. Aust, 1994, Arch. Biochem. Biophys. 312, 511-515). In this work, we investigated protective mechanisms for manganese peroxidase. Oxidation of Mn(II) by manganese peroxidase displayed complex kinetics, which were explained by accumulation of compound III followed by its reactivation by the enzymatically produced Mn(III). Conversion of compound III to ferric enzyme by Mn(III) was not observed for lignin peroxidase or heme propionate-modified recombinant manganese peroxidase, suggesting that Mn(III) may interact with compound III of native manganese peroxidase at a heme propionate to oxidize iron-coordinated superoxide via long-range electron transfer. Additionally, Mn(II) also reactivated compound III. Although this reaction was slower, it could prevent compound III accumulation when excess Mn(II) was present. Another protective mechanism for manganese peroxidase is proposed for insufficient chelator conditions. In contrast to effective Mn(II) chelators, low-affinity ligands supported considerably slower enzyme turnover, and Mn(III) released was more reactive with hydrogen peroxide, resulting in a catalase-type reaction. Reactivation of compound III and catalatic activity may provide biologically relevant mechanisms for protection of manganese peroxidase against suicidal inactivation by hydrogen peroxide under a variety of manganese and oxalate conditions.

Effects of Mn2+ and oxalate on the catalatic activity of manganese peroxidase.

Manganese peroxidase from Phanerochaete chrysosporium is an extracellular heme-containing enzyme known to catalyze the oxidation of Mn2+ to Mn3+ in a reaction requiring oxalate or another appropriate manganese chelator. We have found that the enzyme can also catalyze a manganese-dependent disproportionation of hydrogen peroxide when a manganese chelator is not included. The catalatic activity was observed in the pH range from 3.0 to 8.5, and the apparent second-order rate constant for catalatic reaction was about 2 x 10(5) M-1 s-1 at pH 4.5 to 7.0 at 25 degrees C. Oxalate inhibited oxygen production by increasing the apparent K(m) for Mn2+ for catalatic activity from micromolar to millimolar levels and facilitating peroxidase activity. Catalase-type function was recovered by excess of Mn2+ in the presence of oxalate. We propose that catalatic activity may protect the enzyme from inactivation by hydrogen peroxide in an environment where free oxalate may be limited.

Kinetics of calcium release from manganese peroxidase during thermal inactivation.

It was previously reported that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was susceptible to thermal inactivation because it contains relatively labile Ca2+ ions required for stability and activity [Sutherland and Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. In this work we determined that four Ca2+ ions are present in the enzyme as isolated but this was reduced to 2 mol/mol upon treatment with Ca2+-chelating agents or extensive dialysis of dilute enzyme. One of two relatively tightly bound Ca2+ remaining in the enzyme was released during thermal inactivation at pH 7.2. Inactive enzyme contained one Ca2+ which could be removed in acidic conditions. Inactivation kinetics were biphasic and the rates for the two inactivation steps and the release of Ca2+ during inactivation suggested that the first, faster phase of inactivation was coupled to the removal of Ca2+. The weakly associated Ca2+ normally present in the enzyme did not affect enzyme activity and did not seem to protect the enzyme from thermal inactivation at submicromolar enzyme concentrations. Excess Ca2+ or Mn2+ decreased the rate of the thermal inactivation and Mn2+ stabilized the enzyme more efficiently than Ca2+ at higher temperature. Enzyme stabilization by Mn2+ was proposed to be due to binding of Mn2+ to the Mn2+ substrate binding site. In competition studies, Ca2+ was shown to bind to this site with apparent dissociation constants of 10(-2) and 10(-4) M at pH 4.5 and 7.2, respectively. Moreover, Ca2+ was a poor inhibitor of manganese peroxidase activity at pH 4.5. It is therefore suggested that Ca2+ is absent from the substrate site in physiological conditions but can bind to this site at higher pH and therefore may stabilize the enzyme by binding to both the Mn2+ site and, as previously proposed, to the distal Ca2+ site.

Anti-inflammatory and antishock water-soluble polyesters of glucocorticoids with low level systemic toxicity.

PURPOSE: The objective of this study was to evaluate the pharmacological activity of glucocorticoid hormones incorporated into the structure of water-soluble carbochain polymers via the esterified 21-CH2OH group. METHODS: Polymer analogs of glucocorticoids were prepared by radical copolymerization of 1-vinyl-2-pyrrolidone with cortisol or dexamethasone 21-crotonates and crotonic acid or 2-(diethylamino) ethylcrontonate which served as ionogenic comonomers. Anti-inflammatory, immunosuppressive and catabolic activities for ionogenic tertiary copolymers and previously prepared non-ionogenic binary copolymers were evaluated in standard animal models. The antishock activity of some of the copolymers was evaluated using the "declamping shock" model. RESULTS: Water-soluble tertiary copolymers with a steroid content up to 14 mol% and an intrinsic viscosity up to 0.30 dl/g in ethanol were synthesized. It was shown that the copolymers were stable in aqueous solutions at pH 5.2-7.3. All of the polymers studied suppressed inflammatory reactions at the level of free hormones when administered interperitoneally. The antishock activity was considerably higher compared to free steroids. The copolymers, unlike unmodified glucocorticoids, did not influence the physical development of young animals. They also caused much lower thymus hypotrophy than free hormones. No clear effect of the presence and nature of ionogenic units in copolymers on the pharmacological performance of the copolymers was detected. CONCLUSIONS: The water-soluble polymers bearing glucocorticoid 21-residues in the side chains retain the anti-inflammatory activity of free steroids and exhibit a higher antishock, a lower immunosuppressive and no catabolic effect.