Structure Of Bovine Liver Catalase Research Articles

Au(III) complexes of symmetrical tetradentate Schiff base ligands: Synthesis, characterization, anticancer/antioxidant potency, in silico prediction, and catalase binding properties

Two new gold(III) complexes, [AuL1]Cl3, A, and [AuL2]Cl3, B, were synthesized and characterized. DFT calculations showed square planar geometry for both complexes. Antioxidant results showed that the ability of compounds to inhibit DPPH• is as follows: A > B > L2 > L1. The anticancer assay showed that both complexes have the ability to inhibit the growth of HCT116 colon cancer cell lines, but B shows a relatively better effect than cisplatin. The interaction effects of A and B on the activity and structure of the bovine liver catalase (BLC) were investigated by spectroscopic techniques and molecular docking. Both complexes were able to change the performance and structure of BLC; B has a greater effect on BLC activity, so at concentration of 2 μM, A and B inhibit initial BLC activity by 92.2% and 55.6%, respectively. The interaction mechanism of A and B with BLC was also different; A interacted mostly with van der Waals interactions and hydrogen bonds, but the most important forces for B was hydrophobic interactions. According to docking results, both complexes prefer the domain III of BLC, but B has higher affinity toward BLC than A, which supports the results of spectroscopic and FMO analysis.

A multidisciplinary study for investigating the interaction of an iron complex with bovine liver catalase

Catalase (CAT) is an essential protein protecting the cell from oxidative damage by reactive oxygen species. CAT is a heme enzyme in which iron metal plays a crucial role in catalytic activity. In this research, an iron (II, III) complex ([Fe (bpy)3] [Fe (dipic)2]2.7H2O; dipic−2 = pyridine‐2,6‐ dicarboxylato and bpy = 2,2′‐bipyridine) was used to evaluate its binding interactions with bovine liver catalase (BLC) using spectroscopic and molecular docking methods. The experimental results demonstrated that the catalytic activity of BLC increased slightly and reached 106% of the initial activity. The interactions between Fe complex and BLC led to the quenching of the catalase fluorescence emission via the static quenching mechanism. Thermodynamic parameters demonstrated that the predominant interactions between catalase and Fe complex are hydrogen bond and van der Waals and the process is exothermic and enthalpy driven. The circular dichroism (CD) and synchronous fluorescence results showed that the Fe complex altered the structure and conformation of BLC. It changed the secondary structure of BLC by decreasing α‐helix and β‐sheet content. Also, the experimental data were analyzed using Multivariate Curve Resolution–Alternating Least Square (MCR‐ALS) to the resolution of measured complex spectra and estimate the number of independent chemical species. The docking results in agreement with experimental data showed that the cationic part of the complex with catalase is mainly hydrophobic and van der Waals interactions, and for the anionic part are hydrophobic, van der Waals, and hydrogen bonding.

Industrially important enzyme bovine liver catalase forms amyloid in the presence of 14-4-14 Gemini surfactant at physiological pH

Bovine liver catalase (BLC), a homotetramer (Mr = 28,000), is an industrially and pharmaceutically important enzyme. A cationic Gemini surfactant (tetramethylene-1,4-bis(dimethyltetradecylammonium bromide); 14-4-14) was found to induce aggregation in BLC, as confirmed by Rayleigh light scattering and dynamic light scattering up to ∼150 μM, at a critical micellar concentration (CMC) at physiological pH. On further increasing, the Gemini concentration aggregate disappeared. Secondary and tertiary structural studies confirmed that BLC structure perturbation occurs in two phases: first, loss of both secondary and tertiary structures till the CMC of 14-4-14; and second, recovery of the secondary structure above the CMC, unlike the tertiary structure that showed further solvent accessibility of tryptophan residues, which resulted in unfolding. Moreover, 14-4-14 may have unfolded the enzyme by either directly interacting or opening the hydrophobic patches or engineering solvent shells and reversibly disrupting helical structures until the concentration reaches the CMC. However, above the CMC, with the probable steric hindrance in 14-4-14 active groups due to polymerization, the BLC secondary structure recovered. The thioflavin T fluorescence and transmission electron microscopy results showed that the aggregates were amyloid in nature. Molecular docking analysis also confirmed that 14-4-14 interacted with BLC mainly through hydrophobic interactions, along with a minor contribution of electrostatic interactions. Thus, this study showed how to reverse the surfactant induced BLC amyloid at physiological pH and 298 K, which could be exploited industrially.

Catalytic activity and structural changes of catalase in the presence of Levothyroxine and Isoxsuprine hydrochloride

Two drugs that pregnant women (with hypothyroidism) may use during pregnancy include Isoxsuprine hydrochloride (ISO) and levothyroxine (LEV); ISO to reduce uterine contractions and LEV for the treatment of hypothyroidism. In the current work, we explored the mechanism of binding affinity between the above drugs and antioxidant enzyme Bovine Liver Catalase (BLC). The experimental results confirmed that both drugs could bind with BLC to form drug−BLC complexes but LEV showed a higher binding affinity toward enzyme. The binding constants of LEV–and ISO–BLC were 0.42 × 105 and 0.13 × 104 M−1 at 310 K, respectively. LEV enhanced the catalase activity but the enzymatic activity of BLC reduced gradually in the presence of ISO. Both drugs were able to induce conformational changes in the BLC structure. The results of the molecular docking investigations confirmed the experimental data and showed that the main binding forces in the LEV−BLC and ISO–BLC systems were hydrogen bond and hydrophobic force. The best binding site of both drugs on BLC is located at a cavity among the wrapping domain, N-Terminal arm, and β-barrel.

Molecular docking and inhibitory effects of a novel cytotoxic agent with bovine liver catalase

The synthesis of new metal complexes with fewer side effects than old drugs, such as cisplatin, is the major research pathway of many scientists. In this regard, the study of interactions between the promising complexes and certain targets such as enzymes is necessary. In this work, we reported the synthesis of Zn(II) complex of a novel bidentate Schiff base ligand, SBL, obtained from the condensation of 1-(2-aminoethyl)piperazine and 2,3-butanedione monoxime. The anticancer activities of [Zn(SBL)2]Cl2 complex against HT-116 cancer cell lines were studied by MTT assay and revealed that this complex can induce the cytotoxicity in the human colon cancer cell. The side effects of the prepared complex on the structure and catalytic function of bovine liver catalase (BLC) were evaluated by various spectroscopic and theoretical methods. The enzyme inhibition kinetics showed that Zn(II) complex inhibited catalase function by mixed-type inhibition. UV–Vis spectra, CD spectroscopy, synchronous, and 3D fluorescence confirmed the conformational changes in the structure of BLC in the presence of Zn complex. The fluorescence measurements showed that this complex quenches the intrinsic fluorescence of BLC via static quenching mechanism. The values of binding constants were obtained 2.68 × 104 M−1 and 0.48 × 104 M−1 at 300 and 310 K, respectively. The thermodynamic parameters revealed that the reaction of Zn(II) complex with catalase could proceed spontaneously through van der Waals forces and hydrogen bonds. The molecular docking simulation in the well coherent with kinetics results showed that there is one binding site for Zn(II) complex on BLC and this complex did not directly bind to the catalase active site.

Catalase inhibition by two Schiff base derivatives. Kinetics, thermodynamic and molecular docking studies

Cisplatin and its derivatives, because of their success in treating cancer, continue to be of interest to researchers and an incentive for them to synthesize new transition metal complexes. So, we have explored the effects of two potential anticancer metal complexes which have different metal centers on the function and structure of bovine liver catalase (BLC) by spectroscopic and molecular docking simulation. Kinetics results showed both complexes inhibited BLC through a mixed-type inhibitory mechanism. The molecular docking study in the well coherent with kinetics results confirmed that Pd and Zn complexes did not directly bind into the catalase activity site, but the binding of them into the enzyme cavity influenced the microenvironment of the BLC activity site which resulted in the reduced BLC activity. Results obtained from UV–Vis, circular dichroism (CD) and synchronous fluorescence showed that the structure and conformation of BLC were altered by Zn (II) and Pd(II) complexes. Both complexes changed the secondary structure of BLC by decreasing α-helix and increasing β-sheet content. According to binding constant values (K300 K = 6.31 ± 2.67 × 105 M−1, K310 K = 0.89 ± 0.09 × 105 M−1 for Zn complex and K300 K = 0.40 ± 0.03 × 105 M−1, K310 K = 0.19 ± 0.02 × 105 M−1 for Pd complex), they quenched the intrinsic fluorescence of BLC via a static mechanism. Thermodynamic parameters in agreement with molecular docking results demonstrated that Schiff base complexes could combine with BLC spontaneously through hydrogen bonds and Van der Waals forces. The results of the molecular docking calculations show that the best binding site of both complexes on BLC is located at a cavity among the wrapping domain, N-Terminal arm and β-barrel. All of these results show the relative similarities of zinc and palladium complexes in biological interactions and provides experimental evidence for better understanding the activity mechanism of different anticancer drugs.

How a promising anti-cancer derivative of palladium consisting phen-imidazole ligand affects bovine liver catalase functionality

Promising background of using Nobel metal complexes such as cisplatin in cancer therapy has been emerged as encourage for scientists to design new species with the outlook of finding acceptable alternatives holding less side effects. That’s why study of interactions of promising complexes with special targets such as enzymes seems necessary. As similarity between coordination chemistry of Pd(II) and Pt(II) has been an excuse for scientists to find an alternative to cisplatin, we investigated the biological effects of [Pd(dach)(FIP)](NO3)2 compound containing a novel phen-imidazole ligand, FIP, and diamino cyclohexane (dach) on bovine liver Catalase (BLC) structure and function. Due to UV–vis study, with an increase in palladium compound’s concentration to 30 μM, the enzymatic activity reduced gradually. Afterwards in higher concentrations remained constant at around 65%. Meanwhile Km, kcat and vmax followed an upward trend which confirm mixed inhibitory mechanism.Although based on fluorescence quenching measurements in 25 and 37 °C catalase activity changes slightly, three-dimensional environment surrounding chromophores of the enzyme structure changes considerably in the presence of palladium complex. According thermodynamic parameters of this interaction, it is believed that hydrophobic interactions are influential in the binding process. Furthermore, CD spectroscopy data revealed that Pd(II) complex makes the secondary structure of BLC less stable. Despite remarkable structural changes of BLC due to hydrophobic interactions with ligands, its function was not influenced considerably. The results might expand horizons of medicinal chemists who are looking for metallic compounds as anticancer drugs with fewer side effects.

The inhibitory effect of farnesiferol C against catalase; Kinetics, interaction mechanism and molecular docking simulation

Farnesiferol C (FC) is a natural sesquiterpene coumarin, which includes a widely range of biological activities. In this work, effects of FC on the structure and catalytic function of bovine liver catalase (BLC) was assessed by various spectroscopic and theoretical methods. Kinetic studies showed that FC has a remarkable inhibitory activity on BLC via mixed-type inhibition. The IC50 value as the inhibitory strength of FC was evaluated 1.5μM. Fluorescence spectroscopy, synchronous fluorescence, CD spectroscopy and UV–vis absorption studies revealed conformational changes in the tertiary and secondary structure of BLC as well as the position of the heme group in the presence of different concentrations of FC. Fluorescence studies revealed that FC quenches intrinsic emission of catalase via static quenching process. The binding constants at 298 and 310K were calculated 1.17×105M−1 and 1.0×105M−1, respectively. Thermodynamic data suggested that hydrophobic interactions play a major role in the binding reaction of FC on BLC. Structural studies indicated that the binding FC to the enzyme is responsible for the changes of the percentage of secondary structures' elements especially α-helix. From the simulation data, the role of Arg353 residue in the mechanism of catalase inhibition has been recognized.

Activation of catalase by pioglitazone: Multiple spectroscopic methods combined with molecular docking studies.

Pioglitazone is an important prescription antidiabetic drug with positive roles in controlling high blood sugar in patients with type 2 diabetes. In the present study, we investigated the effects of pioglitazone on the structure and function of bovine liver catalase (BLC) using different spectroscopic and theoretical methods. UV-Vis absorption, fluorescence spectroscopy, synchronous fluorescence, and circular dichroism studies revealed conformational changes in the BLC structure and heme group in the presence of different concentrations of pioglitazone. Kinetic studies indicated that pioglitazone can increase BLC activity by approximately threefold compared with free enzyme. The fluorescence quenching data showed one binding site for pioglitazone, and the binding constants at 298, 304, and 310K were calculated as 5.01×107 M-1 , 5.8×107 M-1 , and 6.6×107 M-1 , respectively. The static type of quenching mechanism was mainly involved in the quenching of intrinsic emission of the enzyme. Thermodynamic data suggested that hydrophobic interactions played a major role in the binding reaction of pioglitazone with BLC. The molecular docking studies indicated that pioglitazone interacts with the cavity in the middle of the β-barrel and wrapping domain of BLC. Thus, pioglitazone can increase catalase activity by changing the BLC structure.

Spectroscopic investigation of Bovine Liver Catalase interactions with a novel phen-imidazole derivative of platinum

Successful clinical experience of using cisplatin and its derivatives in cancer therapy has encouraged scientists to synthesize new metal complexes with the aim of interacting with special targets such as proteins In this regard, biological effects of [Pt(FIP)(Phen)](NO3)2 compound which contains a novel phen-imidazole ligand, FIP, was investigated on bovine liver catalase (BLC) structure and function. Various spectroscopic methods such as UV–visible, fluorescence, and circular dichroism (CD) were applied at two temperatures 25 and 37°C for kinetics and structural studies. As a consequence, the enzymatic activity decreased slightly with increasing the platinum compound’s concentration up to 30 μM and then remained constant at near 80% after this concentration. On the other hand, the fluorescence quenching measurements revealed that despite slight changes in activity, catalase experiences notable alterations in three-dimensional environment around the chromophores of the enzyme structure with increasing platinum complex concentration. Moreover, quenching data showed that BLC has two binding sites for Pt complex and hydrogen bonding interactions play a major role in the binding process. Furthermore, CD spectroscopy data showed that Pt(II) complex induces significant decrease in α-helix content of the secondary structure of BLC, but notable increase in random coil proportion accompanying a slight decrease in β-sheet content. All in all, hydrogen bonding interactions which are mainly involved in the binding process of the novel phen-imidazole compound to BLC significantly alter the protein structure but slightly change its function. This might be a promising outcome for chemotherapists and medicinal chemists to investigate in vivo properties of this novel metal complex with significant binding tendency to a macromolecule in the low concentrations without decreasing its intrinsic function.

Investigation of the binding mechanism and inhibition of bovine liver catalase by quercetin: Multi-spectroscopic and computational study.

Introduction: The study on the side effects of various drugs and compounds on enzymes is a main issue for monitoring the conformational and functional changes of them. Quercetin (3,5,7,3ʹ,4ʹ-pentahydroxyflavone, QUE), a polyphenolic flavonoid, widely found in fruits, vegetables and it is used as an ingredient in foods and beverages. The interaction of bovine liver catalase (BLC) with QUE has been studied in this research by using different spectroscopic methods. Methods: In this work, the interaction of QUE with BLC was investigated using different spectroscopic methods including ultraviolet-visible (UV-vis) absorption, circular dichroism (CD) and fluorescence spectroscopy and molecular docking studies. Results: Fluorescence data at different temperatures, synchronous fluorescence and CD studies revealed conformational changes in the BLC structure in the presence of different concentration of QUE. Also, the fluorescence quenching data showed that QUE can form a non-fluorescent complex with BLC and quench its intrinsic emission by a static process. The binding constant (Ka) for the interaction was 104, and the number of binding site was obtained ~1. The ∆H, ∆S and ∆G changes were obtained, indicating that hydrophobic interactions play a main role in the complex formation. In vitro kinetic studies revealed that QUE can inhibit BLC activity through non-competitive manner. Molecular docking study results were in good agreement with experimental data, confirming only one binding site on BLC for QUE at a cavity among the wrapping domain, threating arm and β-barrel. Conclusion: Inhibition of BLC activity upon interaction with QUE demonstrated that in addition to their beneficial effects, they should not be overlooked for their side effects.

The effect of deep eutectic solvents on catalytic function and structure of bovine liver catalase

Aqueous solutions of reline and glyceline, the most common deep eutectic solvents, were used as a medium for Catalase reaction. By some spectroscopic methods such as UV–vis, fluorescence and circular dichroism (CD) function and structure of Catalase were investigated in aqueous solutions of reline and glyceline. These studies showed that the binding affinity of the substrate to the enzyme increased in the presence of 100mM glyceline solution, which contrasts with reline solution that probably relates to instructive changes in secondary structure of protein. Meanwhile, enzyme remained nearly 70% and 80% active in this concentration of glyceline and reline solutions respectively. In the high concentration of DES solutions, enzyme became mainly inactive but surprisingly stayed in nearly 40% active in choline chloride solution, which is the common ion species in reline and glyceline solvents. It is proposed that the chaotropic nature of choline cation might stop the reducing trend of activity in concentrated choline chloride solutions but this instructive effect is lost in aqueous deep eutectic solvents. In this regard, the presence of various concentrations of deep eutectic solvents in the aqueous media of human cells would be an activity adjuster for this important enzyme in its different operation conditions.

Stabilization of Enzymes Through Encapsulation in Liposomes.

Phospholipid vesicle (liposome) offers an aqueous compartment surrounded by lipid bilayer membranes. Various enzyme molecules have been reported to be encapsulated in liposomes. The liposomal enzyme shows peculiar catalytic activity and selectivity to the substrate in the bulk liquid, which are predominantly derived from the substrate permeation resistance through the membrane. We reported that the quaternary structure of bovine liver catalase and alcohol dehydrogenase was stabilized in liposomes through their interaction with lipid membranes. The method and condition for preparing the enzyme-containing liposomes with well-defined size, lipid composition, and enzyme content are of particular importance, because these properties dominate the catalytic performance and stability of the liposomal enzymes.

Generation of reactive oxygen species via inhibition of liver catalase by oxalli-palladium: A spectroscopic and docking study

In the present study, the side effects of an anti-cancer compound of oxali-palladium on the function and structure of bovine liver catalase (BLC) were investigated using multiple spectroscopic (fluorescence, circular dichroism (CD) and UV–vis) and molecular docking methods. Results of kinetics study showed a noncompetitive inhibition of the enzyme for oxali-palladium. Addition of various concentrations of oxali-palladium caused a gradual reduction in the intrinsic fluorescence emission intensity of BLC due to quenching the fluorescence of BLC via a static type of quenching mechanism. Also addition of oxali-palladium to the enzyme led to increasing content of α-helix and decreasing of β-pleated sheet and random coil structures. The molecular docking study in well coherent with fluorescence spectroscopy illustrated that there is one binding site for oxali-palladium on bovine liver catalase. Docking results confirmed that static quenching is occurred whereas oxali-palladium is located at the distance of Foster theory. Moreover, docking examination in agreement with binding analyzing revealed electrostatic interaction is dominant driving force in oxali-palladium binding to BLC. According above results, it can be concluded that inhibition of BLC by anticancer drug of oxali-palladium increases the content of reactive oxygen species (ROS) which is one of the mechanisms of different anticancer drugs.

Protective effects of aspirin on the function of bovine liver catalase: A spectroscopy and molecular docking study

Amongst all different kinds of analgesic drugs, aspirin (acetylsalicylic acid) is most commonly used to relieve minor pains, reduce fever and to prevent inflammation. To explore the possible side effects of aspirin on the antioxidant defense system of the liver, we analyzed the conformational and functional changes of bovine liver catalase (BLC) in the presence of aspirin, using different spectroscopic techniques including UV–Visible, fluorescence and circular dichroism (CD). No major changes in activity were seen for BLC in the presence of aspirin as observed by kinetics studies. Our results showed that the intrinsic fluorescence of BLC is quenched by aspirin via a static mechanism. Also thermodynamic parameters were calculated showing that van der Waals forces and hydrogen bonds play a major role in this interaction. Moreover, the UV–Vis absorption data in agreement with Trp fluorescence results demonstrated that the microenvironment of Trp residues in BLC is disrupted by aspirin binding. CD results have further shown a significant change in the secondary structure of BLC with an increase in the α-helical content from 24.2% in native protein to 47.4% in BLC-aspirin and a decrease in β-pleated sheets content from 21% in native BLC to 12% upon aspirin addition. The molecular docking results are in well agreement with the experimental data confirming that there exists one binding site for aspirin on BLC at the distance of Forster theory. Additionally, the docking calculations showed that a complex was formed between aspirin and BLC consistent with the data on the intrinsic fluorescence intensity of BLC based on the static quenching mechanism. Finally, all the results from this study have indicated that the addition of aspirin leads to the unfolding of the three-dimensional structure of BLC and can be considered as the side effect of this drug on the antioxidant system of the liver. It might be suggested that in the presence of high concentrations of H2O2, aspirin can protect BLC activity through a mechanism in which the inactive form of the enzyme (compound II) is reduced to the active form.

Inhibitory effects of deferasirox on the structure and function of bovine liver catalase: a spectroscopic and theoretical study

Deferasirox (DFX), as an oral chelator, is used for treatment of transfusional iron overload. In this study, we have investigated the effects of DFX as an iron chelator, on the function and structure of bovine liver catalase (BLC) by different spectroscopic methods of UV–visible, fluorescence, and circular dichroism (CD) at two temperatures of 25 and 37 °C. In vitro kinetic studies showed that DFX can inhibit the enzymatic activity in a competitive manner. KI value was calculated 39 nM according to the Lineweaver–Burk plot indicating a high rate of inhibition of the enzyme. Intrinsic fluorescence data showed that increasing the drug concentrations leads to a significant decrease in the intrinsic emission of the enzyme indicating a significant change in the three-dimensional environment around the chromophores of the enzyme structure. By analyzing the fluorescence quenching data, it was found that the BLC has two binding sites for DFX and the values of binding constant at 25 and 37 °C were calculated 1.7 × 107 and 3 × 107 M−1, respectively. The static type of quenching mechanism is involved in the quenching of intrinsic emission of enzyme. The thermodynamic data suggest that hydrophobic interactions play a major role in the binding reaction. UV–vis spectroscopy results represented the changes in tryptophan (Trp) absorption and Soret band spectra, which indicated changes in Trp and heme group position caused by the drug binding. Also, CD data represented that high concentrations of DFX lead to a significant decreasing in the content of β-sheet and random coil accompanied an increasing in α-helical content of the protein. The molecular docking results indicate that docking may be an appropriate method for prediction and confirmation of experimental results and also useful for determining the binding mechanism of proteins and drugs. According to above results, it can be concluded that the DFX can chelate the Fe(III) on the enzyme active site leading to changes in the function and structure of catalase which can be considered as a side effect of this drug and consequently has an important role in hepatic complications and fibrosis.

Structure of catalase determined by MicroED.

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

Human Erythrocyte Catalase: 2-D Crystal Nucleation and Production of Multiple Crystal Forms

Negatively stained electron microscope images are presented, showing the nucleation of two-dimensional (2-D) crystals of human erythrocyte catalase produced on mica by the negative staining-carbon film technique. Examples of the formation of partially ordered 2-D arrays and more ordered 2-D crystals are shown and the conditions required for the production of large well-ordered 2-D crystals discussed. The structural transformation of one flexuous 2-D paracrystal into a p2 2-D crystal is considered. The crystallographic 2-D image average of this p2 crystal form is presented (lattice parameters a = 9.0 nm, b = 18.6 nm; γ= 90-8°). It is shown that transmission electron microscopy provides the possibility of defining 2-D crystal nucleation, growth of intermediate forms, and low-resolution crystallographic structural analysis of 2-D crystals of human erythrocyte catalase. Comparison of the various electron microscopical negatively stained images with the peptide backbone of the X-ray structure of bovine liver catalase at different tilt and rotation positions correlates with and emphasizes the multiple intermolecular contacts and orientations that can be adopted by human erythrocyte catalase, leading to various 2-D arrays and 2-D crystals. Alignment of the surface groups involved in the protein-protein interactions that occur during 2-D crystal nucleation and crystal growth may ultimately be determined. From this approach, when taken together with detailed consideration of protein-solvent and protein-solute electrostatic interactions in solution, and at the fluid-air interface, it is considered that a more general theory of crystal nucleation and growth may eventually emerge.

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.

Catalase: a tetrameric enzyme with four tightly bound molecules of NADPH.

Catalases (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) from many species are known to be tetramers of 60,000-dalton subunits, with four heme groups per tetramer. Previous authors have determined the amino acid sequence and three-dimensional structure of bovine liver catalase. Studies of the regulation of the pentose phosphate pathway led the present authors to a search for proteins that bind NADP+ and NADPH in human erythrocytes. An unexpected result of that search was the finding that a major reservoir of bound NADPH in human erythrocytes is catalase. Each tetrameric molecule of human or bovine catalase contains four molecules of tightly bound NADPH. The binding sites have the relative affinities NADPH greater than NADH greater than NADP+ greater than NAD+. NADPH does not seem to be essential for the enzymic conversion of H2O2 to O2 and water but does provide protection of catalase against inactivation by H2O2.