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Catalase from acatalasemic dog liver was purified to homogeneity and its properties were compared with those of normal dog liver catalase. The purified acatalasemic and normal dog liver catalases were found to have the same molecular weight (230,000 Da) and isoelectric point (pI: 6.0-6.2) and both enzymes contained four hematins per molecule. The catalytic activity of catalase from acatalasemic dog was normal. Furthermore, there was no difference between the acatalasemic and normal dog catalases in the binding affinity to NADPH (apparent Kd: 0.11-0.12 microM) and in the sensitivity to oxidative stress by hydrogen peroxide, the normal substrate of catalase. The acatalasemic dog enzyme was stable only in a narrow pH range (pH 6-9) although the normal enzyme was stable in a wide pH range (pH 4-10). Acatalasemic dog liver catalase also showed a slight low thermal stability at 37 degrees C and the heat-lability was remarkable at 45 degrees C, compared to the normal dog enzyme. These results indicated that the acatalasemic dog catalase is catalytically normal although it is associated with an unstable molecular structure.
As Mycobacterium leprae proliferate inside macrophages, it has been speculated that catalase encoded by katG may protect the bacilli from deleterious effects of peroxide generated from the macrophage and may also play a crucial role in the survival of M. leprae in vivo. However, unlike that of M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implicating that isoniazid, which is activated to a potent tuberculocidal agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. These results raise a question as to how M. leprae avoids H202-mediated killing inside macrophages. To understand the survival of M. leprae in macrophages, the present study attempted to detect catalase-like activity in M. leprae. Catalase-like activity was found in M. leprae cell lysate by the diaminobenzidine (DAB) staining method with non-denaturing polyacrylamide gel electrophoresis. An ammonium sulphate precipitation study revealed that the catalase-like activity was precipitable with 80% ammonium sulphate. The effect of isoniazid (INH) on M. leprae growth was also tested by RT-PCR and radiorespirometric assay to examine catalase-like activity in M. leprae, because INH was activated by catalase. It was found that the viability of M. leprae was decreased at a concentration of 20 microg/ml by radiorespirometric assay and it was inhibited at higher concentrations as determined by RT-PCR. These data suggest that a catalase-like activity other than that encoded by katG is present in M. leprae.
Intracellular killing of catalase-positive bacteria by murine resident macrophages requires the presence of extracellular serum, whereas killing of catalase-negative bacteria can occur in the absence of serum. To find out whether the intracellular killing of bacteria by rIFN-gamma-activated macrophages also requires serum stimulation, we investigated the handling of ingested catalase-negative and -positive Listeria monocytogenes by peritoneal macrophages of normal Swiss mice and mice injected i.p. with 1 x 10(4) U rIFN-gamma 18 h earlier. In the absence of extracellular serum, rIFN-gamma-activated macrophages killed ingested catalase-negative Listeria more efficiently (P < 0.01) than normal resident macrophages. Maximal killing of catalase-negative bacteria by rIFN-gamma-activated macrophages required an extracellular serum concentration of only 1.0 to 2.5% compared with the 10% needed by normal macrophages. No differences were observed in the rates of intracellular killing of catalase-positive Listeria by rIFN-gamma-activated and normal resident macrophages: both populations of macrophages required 10% extracellular serum for maximal killing of these bacteria, and killing was minimal in the absence of serum. The rIFN-gamma-activated macrophages displayed enhanced O2-consumption after stimulation with phorbol myristate acetate and heat-killed Listeria compared with macrophages from normal mice. These findings indicate that, under suboptimal stimulation by extracellular serum, rIFN-gamma enhances the intracellular killing of catalase-negative Listeria which lack endogenous catalase acting as a scavenger of reactive oxygen intermediates. The mechanism underlying the enhancement is probably the amplification of the respiratory burst by IFN-gamma.
Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamide adenine dinucleotide peroxidase during late growth in the glucose-exhausted medium. The catalase-positive strain and the catalase-negative strain tested both possessed low but readily detectable levels of superoxide dismutase throughout growth. The growth responses are discussed in terms of the presence of enzymes which would allow the cells to remove potentially damaging reduction products of O2.
We have converted a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase using DNA shuffling and error-prone PCR. A triple mutant, R47H/R356C/D374N, that showed significantly reduced catalase activity and increased peroxidase activity was identified by screening mutant libraries. When single mutant--R47H, R356C and D374N--were generated by site-directed mutagenesis, conserved Arg-47, located on the distal side of the prosthetic heme group in the superfamily of typical catalases, was found to be responsible for the conversion of catalase to catalase-peroxidase. To further clarify the role of Arg-47, arginine was replaced with different amino acids--alanine, lysine, aspartic acid, glutamic acid, glutamine, phenylalanine, tryptophan and tyrosine--and the mutant enzymes were assayed. All of the arginine mutants had increased peroxidase activity coupled with reduced catalase activity. Among these mutants, R47W exhibited the highest peroxidase activity, while R47E and R47Q not only had increased peroxidase activity but also retained relatively high catalase activity. These results suggest that tryptophan plays a key role in the catalytic mechanism of the peroxidase reaction and that glutamic acid and glutamine facilitate both catalatic and peroxidatic reactions.
The bacterium Klebsiella pneumoniae synthesizes three different types of catalase: a catalase-peroxidase, a typical catalase and an atypical catalase, designated KpCP, KpT and KpA, respectively (Goldberg, I. and Hochman, A. (1989) Arch. Biochem. Biophys. 268, 124-128). KpCP, but not the other two enzymes, in addition to the catalatic activity, catalyzes peroxidatic activities with artificial electron donors, as well as with NADH and NADPH. Both KpCP and KpT are tetramers, with heme IX as a prosthetic group, and they show a typical high-spin absorption spectrum which is converted to low-spin when a cyanide complex is formed. The addition of dithionite to KpCP causes a shift in the absorption maxima typical of ferrous heme IX. KpCP has a pH optimum of 6.3 for the catalatic activity and 5.2-5.7 for the peroxidatic activity, and relatively low 'Km' values: 6.5 mM and 0.65 H2O2 for the catalatic and peroxidatic activities, respectively. The activity of the catalase-peroxidase is inhibited by azide and cyanide, but not by 3-amino-1,2,4-triazole. KpT has wide pH optimum: 5-10.5 and a 'Km' of 50 mM H2O2, it is inhibited by incubation with 3-amino-1,2,4-triazole and by the acidic forms of cyanide and azide. A significant distinction between the typical catalase and the catalase-peroxidase is the stability of their proteins: KpT is more stable than KpCP to H2O2, temperature, pH and urea.
3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.
Catalysis of hydrogen peroxide dismutation by the dimanganese catalase from Thermus thermophilus has been measured and found to obey Michaelis-Menton kinetics with no evidence for substrate inhibition at concentrations up to 0.45 M H2O2. Comparison among three dimanganese catalases (Thermus thermophilus, Thermoleophilium album, and Lactobacillus plantarum) reveals that their apparent second-order rate constants, Kcat/Km, differ by at most a factor of 5, even though the individual kinetic constants differ by as much as a factor of 20. This similarity suggests that all three enzymes may have the same rate-determining step. For T. thermophilus catalase we find that kcat/Km approximately kbi, the bimolecular rate constant at limiting substrate concentrations. Thus, the rate of the rate-determining step is unaltered over the entire range of substrate concentrations, unlike T. album and L. plantarum catalases where substrate inhibition has been reported. Comparison to structurally characterized dimanganese complexes and dimetalloproteins (arginase, hemerythrin), which are functional, albeit kinetically slow, catalase mimics, reveals that high catalase activity correlates with a greater number of stronger sigma-ligand donors like anionic carboxylatos vs neutral histidines that stabilize the oxidized Mn2(III,III) state over reduced Mn2(II,II). A critical feature for enzymatic functionality in vivo is suppression of one-electron chemistry leading to formation of the mixed-valence forms, Mn2(III,IV) and Mn2(II,III), which are kinetically inactive or precursors to inactive species, respectively. Evidence is presented from model compounds suggesting that the mu-carboxylato bridge between Mn ions in catalase may play the key role in suppressing formation of these detrimental oxidation states through destabilization of these one-electron redox processes.
The N-terminal sequence of a protein, originally described as an adhesin of Helicobacter pylori, was used in an oligonucleotide-based screening procedure of an H. pylori plasmid library in Escherichia coli. Five independent plasmid clones were isolated, all mapping to the same chromosomal region and encoding the H. pylori catalase. The gene, designated katA, comprises 1,518 nucleotides and encodes a putative protein of 505 amino acids with a predicted Mr of 58,599. A second open reading frame, orf2, encoding a putative 32,715-Da protein of unknown function, follows katA. The transcriptional start site of katA mRNA was determined, but no typical consensus promoter sequence was present. A potential binding site for the Fur protein is located upstream of katA. When introduced into the catalase-deficient E. coli double-mutant UM255, the cloned gene readily complemented E. coli for catalase activity. H. pylori KatA is highly homologous to catalases in both prokaryotes and eukaryotes, with the highest homology being shown to Bordetella pertussis (64.9%), Bacteroides fragilis (59.8%), and Haemophilus influenzae (57.9%) catalases. Transposon insertion mutants were generated in three independent H. pylori strains by TnMax5-mediated transposon shuttle mutagenesis. In contrast to the wild-type strains, no significant catalase-specific enzymatic activity could be detected in the mutant strains, consistent with the fact that no additional katA-homologous gene copies were found in the H. pylori chromosome. No significant difference between wild-type and mutant strains for binding to epithelial cells was apparent, suggesting that KatA is not involved in H. pylori adhesion. The cloning and genetic characterization of katA are essential steps for further investigation of the role of catalase in the defense of H. pylori against oxygen-dependent killing mechanisms by polymorphonuclear granulocytes, a process not well understood for this chronically persisting pathogen.
Catalase enzyme was purified from human erythrocytes. The modified procedure of Mörikofer-Zwez et. al. [Eur. J. Biochem. 11: 49-57, 1969] yielded erythrocyte catalase with high specific activity and with one band on SDS polyacrylamide gel. Its other characteristics (isoelectric point; E405/280, E1%1cm at 280 nm and 405 nm) were in agreement with previously described findings. The results obtained for molecular mass, electrophoretic mobility, chromatographic behaviour on CM-Sepadex gel, addition test, and change of electrophoretic mobility in human serum showed differences between human erythrocyte catalase and bovine liver catalase. These results suggest that human erythrocyte catalase and bovine liver catalase are two distinct catalase forms.
To investigate the importance of catalase as a protecting enzyme against oxidative damage in phagocytic leukocytes, we have tested the functional capacity of neutrophils from two individuals homozygous for Swiss-type acatalasemia and from two individuals heterozygous for this deficiency. In the former cells, 25-30% of residual activity of catalase was present. In the latter cells, the values were close to normal. Chemotaxis towards casein, release of lysosomal enzymes and hydrogen peroxide during phagocytosis of zymosan, and intracellular killing of Staphylococcus aureus were normal in all cells tested. Inhibition of heme enzymes with azide (2 mM) enhanced the respiration and hexose monophosphate shunt activity of normal, but not of homozygous acatalasemic, neutrophils. This indicates that the enhancement in normal cells is, at least in part, due to catalase inhibition. After 15 min preincubation with an H(2)O(2)-generating system (glucose plus glucose oxidase), the respiratory response to zymosan phagocytosis was strongly depressed in the homozygous acatalasemic and in normal, azide-treated neutrophils, but not in normal, untreated cells. Under these conditions, the release of lysosomal enzymes was depressed and that of lactate dehydrogenase enhanced, in catalase-deficient and in catalase-inhibited, but not in normal, neutrophils. During prolonged incubation with the H(2)O(2)-generating system (30-60 min), the reduction level of intracellular glutathione remained high and the hexose monophosphate shunt continued to operate normally in all cells tested. Thus, although the function of neutrophils without catalase activity was depressed by extracellular hydrogen peroxide, the H(2)O(2) degradation via the glutathione redox system remained operative. The results indicate that the glutathione redox system by itself efficiently protects phagocytosing neutrophils against their own oxidative products. During heavy external oxidative stress, however, both catalase and the glutathione redox system are needed for adequate protection.
In order to characterize the genetic defect in Japanese-type acatalasemia, we examined catalase synthesis in the cultured fibroblasts isolated from a patient suffering from this disease. The transcript of the mutant catalase gene was also analyzed by Northern blotting. It was demonstrated that the severe reduction in catalase protein in the acatalasemic patient is due to the defective synthesis of the enzyme caused by the lack of catalase mRNA. Using Southern blotting, we did not detect large deletion, insertion or rearrangement in the mutant catalase gene as compared with the normal one. We conclude that Japanese-type acatalasemia in this study is resulted by a small mutation in the non-coding region of catalase gene which affects some step in the catalase mRNA metabolism.
Phanerochaete chrysosporium uses hydrogen peroxide as a substrate in its ligninolytic phase. To determine how the fungus protects itself against detrimental effects of reactive oxygen species, catalase activity was examined. The fungus produced up to four different catalase isozymes, Cat A to D. Isozyme CatC was the dominant activity in all growth conditions. A minor catalase, CatD, was apparent in low-carbon culture and upon exposure of mycelium grown on high-nitrogen medium with 5-50 mm of hydrogen peroxide. In low-nitrogen culture, an increase in catalase-specific activity preceded the onset of ligninolysis by 3 days. In low-carbon culture, catalase was produced at an even higher level without development of ligninolysis. Thus, catalase activity was not coordinately produced with ligninolytic metabolism.
Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.
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.
A mutant of the methanol-utilizing yeast Hansenula polymorpha defective in catalase was isolated. It lacks the ability to grow on methanol as the sole source of carbon and energy due to a loss of peroxisomal function that is required for the dissimilation and assimilation of this substrate. Growth of the mutant on glucose or glycerol was not impaired. Transformation of mutant cells with the gene coding for catalase A from Saccharomyces cerevisiae [Cohen, G., Fessl, F., Traczyk, J., Rytka, J. & Ruis, H. (1985) Mol. Gen. Genet. 200, 74-79] conferred constitutive expression of catalase activity. When the gene was placed under control of the regulatory methanol oxidase promoter from H. polymorpha, high levels of activity subject to glucose repression were obtained. In both cases efficient targeting of catalase A to the heterologous peroxisomes and assembly into an active form could be demonstrated. Concomitantly, growth on methanol was restored in the transformed mutant. The results are in line with a high conservation of transport signals on peroxisomal proteins. Expression of a cytosolic catalase in H. polymorpha did not confer the ability to grow on methanol. Therefore, proper localization of the catalase activity is a prerequisite for peroxisomal function.
Two of the five multiple forms of beef liver catalase have been extensively studied using biochemical and biophysical analysis techniques. The two molecules, cat I and cat V, have different isoelectric points (pH 6.55 and 5.6), different surface charges (25.8 and 32.7 elementary charges) and display different numbers of primary amino groups on their surfaces. The numbers of tyrosine residues on the surfaces of the two molecules are also different (16 and 10 at pH 10). Since the two forms of catalase can be interconverted, the described changes may be caused by conformational changes of the four protein subunits within the molecule. This mobility of the polypeptide chains is also demonstrated by the different absorption spectra below 390 nm. Using small-angle X-ray scattering, the radii and the volumes of the two catalase forms were shown to be different (cat 1 is smaller than cat V). All five multiple forms of beef liver catalase are concormers of the molecule. A model for the quaternary sturcture of the beef liver catalase are conformers of the molecule. A model for the quaternary structure of the beef liver catalase molecule is suggested. It consists of a regular configuration of four prolate rotational ellipsoids (semiaxes: a = 52 A, b = c = 21 A) in close contact in which the nearest neighbour subunits are shifted by 37 A parallel to each other. Thus the height of the complete molecule is 141 A and the diameter 94 A.