The catalase activity of Nalpha-acetyl-microperoxidase-8.

Nalpha-Acetylated microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for peroxidases. We report here that Ac-MP-8 catalyzes catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The catalase activity of Ac-MP-8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The Km and kcat of the decomposition of hydrogen peroxide (H2O2) catalyzed by Ac-MP-8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (kcat/Km) of Ac-MP-8 in catalase-type reaction of H2O2 is 100.2,/m/s, which is 5- to 12- and 50- to 100-fold less than those of MPs in cytochrome P450-type reaction of aniline/H2O2 and peroxidase-type reaction of o-methoxyphenol/H2O2, respectively. These results indicate that Ac-MP-8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroxidase-type > cytochrome P450-type > catalase-type reactions. Comparisons of the enzyme activities of Ac-MP-8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.     

The molecular symmetry of bovine liver catalase.

A rotation function study of bovine liver catalase at 10 Å resolution has shown the enzyme to have at least one 2-fold axis, although a molecular symmetry of 222 is likely and the molecular point group 4 is possible. The orientation of the molecular axes with respect to the crystallographic axes has also been determined.     

The mechanism of catalase action. I. Steady-state analysis

1.

1. Tritium-labeled cholic acid was prepared by biosynthesis in the rat.     

The chemical modification of beef liver catalase. V. Ethoxyformylation of histidine and tyrosine residues of catalase with diethylpyrocarbonate.

In order to elucidate the possible roles of histidine and tyrosine residues of catalase [EC 1.11.1.6] in maintaining the quaternary structure and catalatic activity, diethylpyrocarbonate modification experiments were carried out. A method for the estimation of N-ethoxyformyl (EF)-His at pH 5--7 and of O-ethoxyformyl (EF)-Tyr in alkaline solution by measuring A 242 nm (ximM = 3.2) and A278 nm (ximM = 1.16), respectively, was developed. The formation of EF-His and EF-Tyr was an electrophilic reaction and was dependent on pH, exhibiting pK values of 6.8 and 9.9, respectively. The maximal yield of EF-His at pH 6.0 was 49% of the total histidine content, but no inactivation nor unfolding of the enzyme was observed. The formation of 12 EF-Tyr residues per mole of catalase at pH 8.1 did not cause any inactivation, but the formation of 8 more EF-Tyr residues at pH 8.9 resulted in both inactivation and unfolding. Nearly complete inactivation and partial splitting of catalase were observed when 43-46 EF-Tyr residues per mole were produced at pH 10.0. More EF-His residues were formed by the reaction of diethyl pyrocarbonate with cyanoethylated (CE)-catalase monomer (subunit) than with CE-catalase tetramer. The CE-catalase tetramer and monomer were extensively O-ethoxyformylated, reaching 100% EF-Tyr formation. These results indicate that a half of the histidine residues may lie outside the protein core and that three-quarters of the tyrosine residues are probably in the protein core of the enzyme. The production of 2--3 EF-Tyr residues per mole of the monomer by ethoxyformylation at pH 7.0 was accompanied by a decrease in the magnitude of the Soret peak. A possible interaction of those tyrosine residues with porphyrin of the heme group is discussed.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

The reaction of Aspergillus niger catalase with methyl hydroperoxide.

The formation of Compound I from Aspergillus niger catalase and methyl hydroperoxide (CH3OOH) has been investigated kinetically by means of rapid-scanning stopped-flow techniques. The spectral changes during the reaction showed distinct isobestic points. The second-order rate constant and the activation energy for the formation of Compound I were 6.4 x 10(3) M-1s-1 and 10.4 kcal.mol-1, respectively. After formation of Compound I, the absorbance at the Soret peak returned slowly to the level of ferric enzyme with a first-order rate constant of 1.7 x 10(-3) s-1. Spectrophotometric titration of the enzyme with CH3OOH indicates that 4 mol of peroxide react with 1 mol of enzyme to form 1 mol of Compound I. The amount of Compound I formed was proportional to the specific activity of the catalase. The irreversible inhibition of catalase by 3-amino-1,2,4-triazole (AT) was observed in the presence of CH3OOH or H2O2. The second-order rate constant of the catalase-AT formation in CH3OOH was 3.0 M-1 min-1 at 37 degrees C and pH 6.8 and the pKa value was estimated to be 6.10 from the pH profile of the rate constant of the AT-inhibition. These results indicate that A. niger catalase forms Compound I with the same properties as other catalases and peroxidases, but the velocity of the Compound I formation is lower than that of the others.     

The catalase activity of ferrihaems

1. The variation of the specific stoicheiometric catalatic activity of proto- and deuteroferrihaem with total ferrihaem concentration has been studied at 25 degrees C over a wide range of pH. For deuteroferrihaem the results imply that only monomeric ferrihaem species contribute significantly to the catalatic activity. Protoferrihaem is more highly dimerized in solution and, in this system, contributions to the catalatic activity from both monomeric and dimeric ferrihaem species were observed. The ratio of the specific activity of protoferrihaem monomer to that of dimer varied from approximately 20 at pH7 to 5x10(4) at pH12.2. 2. The specific activity of protoferrihaem monomer closely resembles that of deuteroferrihaem monomer, both in magnitude and pH-dependence. In both cases the activity is inversely proportional to [H(+)]. In contrast, the activity of catalase is independent of pH in the range 5-10. At pH13 the activity of ferrihaem monomer becomes equal to the maximal activity of catalase. The results are in good agreement with those reported by Brown et al. (1970b) and provide support for the assumptions upon which this previous analysis relied. 3. Information from the literature concerning the catalatic activity and dimerization of the iron(III) complex of 4,4',4',4'-tetrasulphophthalocyanine (Waldmeier & Sigel, 1971; Sigel et al., 1971) have been re-analysed. The results imply that both the monomeric and dimeric complexes contribute to catalatic activity and these activities closely resemble those of the corresponding protoferrihaem species.     

The active center of catalase

The refined structure of beef liver catalase (I. Fita, A. M. Silva, M. R. N. Murthy & M. G. Rossmann, unpublished results) is here examined with regard to possible catalytic mechanisms. The distal side of the deeply buried heme pocket is connected with the surface of the molecule by one (or possibly two) channel. The electron density representing the heme group, in each of the two crystallographically independent subunits, is consistent with degradation of the porphyrin rings. The heme group appears to be buckled, reflecting the high content of bile pigment in liver catalase. The spatial organization on the proximal side (where the fifth ligand of the iron is located) shows an elaborate network of interactions. The distal side contains the substrate pocket. The limited space in this region severely constrains possible substrate positions and orientations. The N delta atom of the essential His74 residue hydrogen bonds with O gamma of Ser113, which in turn hydrogen bonds to a water molecule associated with the propionic carbonylic group of pyrrole III. These interactions are also visible in the refined structure of Penicillium vitale catalase (B. K. Vainshtein, W. R. Melik-Adamyan, V. V. Barynin, A. A. Vagin, A. I. Grebenko, V. V. Borisov, K. S. Bartels, I. Fita, & M. G. Rossmann, unpublished results). Model building suggests a pathway for a catalase mechanism (compound I formation, as well as catalatic and peroxidatic reactions). There are some similarities in compound I formation of catalase and cytochrome c peroxidase.     

The grid sectioning technique: a study of catalase platelets.

The grid sectioning technique has been used to obtain the two missing principal axis projections of orthorhombic catalase platelets and to measure directly the unit cell c-value. The negatively stained platelets have a unit cell c-dimension of half that proposed by Unwin (1975) from powder X-ray diffraction. The precision of the grid sectioning technique in positioning sections along a specimen axis shows that the growth fault lines usually observed on negatively stained catalase platelets are rows of missing molecules filled with stain. From these sections conclusions are drawn concerning the action of negative stain on a specimen, the microtomy process, and the specimen/supporting film interaction. Finally the value of microtomy for detailed structural analysis of biological objects is emphasized.