Peroxidase-Like Catalytic Activity of Ag3PO4 Nanocrystals Prepared by a Colloidal Route

Nearly monodispersed Ag₃PO₄ nanocrystals with size of 10 nm were prepared through a colloidal chemical route. It was proven that the synthesized Ag₃PO₄ nanoparticles have intrinsic peroxidase-like catalytic activity. They can quickly catalyze oxidation of the peroxidase substrate 3, 3, 5, 5-tetramethylbenzidine (TMB) in the presence of H₂O₂, producing a blue color. The catalysis reaction follows Michaelis-Menten kinetics. The calculated kinetic parameters indicate a high catalytic activity and the strong affinity of Ag₃PO₄ nanocrystals to the substrate (TMB). These results suggest the potential applications of Ag₃PO₄ nanocrystals in fields such as biotechnology, environmental chemistry, and medicine.     

Magnetosomes extracted from Magnetospirillum magneticum strain AMB-1 showed enhanced peroxidase-like activity under visible-light irradiation

Magnetosomes are intracellular structures produced by magnetotactic bacteria and are magnetic nanoparticles surrounded by a lipid bilayer membrane. Magnetosomes reportedly possess intrinsic enzyme mimetic activity similar to that found in horseradish peroxidase (HRP) and can scavenge reactive oxygen species depending on peroxidase activity. Our previous study has demonstrated the phototaxis characteristics of Magnetospirillum magneticum strain AMB-1 cells, but the mechanism is not well understood. Therefore, we studied the relationship between visible-light irradiation and peroxidase-like activity of magnetosomes extracted from M. magneticum strain AMB-1. We then compared this characteristic with that of HRP, iron ions, and naked magnetosomes using 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate in the presence of H₂O₂. Results showed that HRP and iron ions had different activities from those of magnetosomes and naked magnetosomes when exposed to visible-light irradiation. Magnetosomes and naked magnetosomes had enhanced peroxidase-like activities under visible-light irradiation, but magnetosomes showed less affinity toward substrates than naked magnetosomes under visible-light irradiation. These results suggested that the peroxidase-like activity of magnetosomes may follow an ordered ternary mechanism rather than a ping–pong mechanism. This finding may provide new insight into the function of magnetosomes in the phototaxis in magnetotactic bacteria.     

Rapid biological synthesis of platinum nanoparticles using <Emphasis Type="Italic">Ocimum sanctum</Emphasis> for water electrolysis applications

The leaf extract of Ocimum sanctum was used as a reducing agent for the synthesis of platinum nanoparticles from an aqueous chloroplatinic acid (H₂PtCl₆·6H₂O). A greater conversion of platinum ions to nanoparticles was achieved by employing a tulsi leaf broth with a reaction temperature of 100 °C. Energy-dispersive absorption X-ray spectroscopy confirmed the platinum particles as major constituent in the reduction process. It is evident from scanning electron microscopy that the reduced platinum particles were found as aggregates with irregular shape. Fourier-transform infrared spectroscopy revealed that the compounds such as ascorbic acid, gallic acid, terpenoids, certain proteins and amino acids act as reducing agents for platinum ions reduction. X-ray diffraction spectroscopy suggested the associated forms of platinum with other molecules and the average particle size of platinum nanoparticle was 23 nm, calculated using Scherer equation. The reduced platinum showed similar hydrogen evolution potential and catalytic activity like pure platinum using linear scan voltammetry. This environmentally friendly method of biological platinum nanoparticles production increases the rates of synthesis faster which can potentially be used in water electrolysis applications.     

纳米酶在疾病治疗中的研究与应用

纳米酶是一种新型的具有类酶活性的纳米颗粒人工酶,在生物检测、抗炎、抗氧化损伤和癌症治疗等疾病诊断和治疗领域展现出良好的应用前景。本文总结了具有不同类酶活性的纳米酶在疾病诊治中的应用,并对影响纳米酶活性的主要影响因素进行了阐述,将使相关研究人员更好地了解纳米酶的发展现状,并提供后续研究的相关线索。     

Intrinsic enzyme-like activity of magnetite particles is enhanced by cultivation with Trichoderma guizhouense

Fungal–mineral interactions can produce large amounts of biogenic nano-size (~ 1–100 nm) minerals, yet their influence on fungal physiology and growth remains largely unexplored. Using Trichoderma guizhouense NJAU4742 and magnetite (Mt) as a model fungus and mineral system, we have shown for the first time that biogenic Mt nanoparticles formed during fungal–mineral cultivation exhibit intrinsic peroxidase-like activity. Specifically, the average peroxidase-like activity of Mt nanoparticles after 72 h cultivation was ~ 2.4 times higher than that of the original Mt. Evidence from high resolution X-ray photoelectron spectroscopy analyses indicated that the unique properties of magnetite nanoparticles largely stemmed from their high proportion of surface non-lattice oxygen, through occupying surface oxygen-vacant sites, rather than Fe redox chemistry, which challenges conventional Fenton reaction theories that assume iron to be the sole redox-active centre. Nanoscale secondary ion mass spectrometry with a resolution down to 50 nm demonstrated that a thin (< 1 μm) oxygen-film was present on the surface of fungal hyphae. Furthermore, synchrotron radiation-based micro-FTIR spectra revealed that surface oxygen groups corresponded mainly to organic OH, mineral OH and carbonyl groups. Together, these findings highlight an important, but unrecognized, catalytic activity of mineral nanoparticles produced by fungal–mineral interactions and contribute substantially to our understanding of mineral nanoparticles in natural ecosystems.     

Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase

ABSTRACT

This work shows the effects of exposure to an electromagnetic field at 900?MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry.     

High-affinity binding and catalytic activity of His/Tyr-based sequences: Extending heme-regulatory motifs beyond CP

Background & motivationPeptides and proteins can interact with heme through His, Tyr, or Cys in heme-regulatory motifs (HRMs). The Cys-Pro dipeptide is a well investigated HRM, but for His and Tyr such a distinct motif is currently unknown. In addition, many heme-peptide complexes, such as heme-amyloid β, can display a peroxidase-like activity, albeit there is little understanding of how the local primary and secondary coordination environment influences catalytic activity. We thus systematically evaluated a series of His- and Tyr-based peptides to identify sequence features for high-affinity heme binding and their impact on the catalytic activity of heme.MethodsWe employed solid-phase peptide synthesis to produce 58 nonapeptides, which were investigated by UV/vis, resonance Raman, and 2D NMR spectroscopy. A chromogenic assay was used to determine the catalytic activity of the heme-peptide complexes.ResultsHeme-binding affinity and binding mode were found to be dependent on the coordinating amino acid and spacer length between multiple potential coordination sites in a motif. In particular, HXH and HXXXH motifs showed strong heme binding. Analysis of the peroxidase-like activity revealed that some of these peptides and also HXXXY motifs enhance the catalytic activity of heme significantly.ConclusionsWe identify HXH, HXXXH, and HXXXY as potential new HRMs with functional properties. Several peptides displayed a strikingly high peroxidase-like activity.General significanceThe identification of HRMs allows to discover yet unknown heme-regulated proteins, and consequently, enhances our current understanding of pathologies involving labile heme.     

Formation of a bioconjugate composed of hemin, smectite, and quaternary ammonium chloride that is soluble and active in hydrophobic media

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.     

马利亚霉菌(Mariannaeasp.) HJ合成金属硒化物及机理研究

[背景] 金属硒化物因其优异的光电和催化特性,近年来在半导体、电化学及抗癌等领域成为了研究热点。相较于传统的化学还原法,生物合成金属硒化物具有环境友好、耗能较低等优势。然而,目前有关生物合成金属硒化合物的微生物资源较少且相关合成机理尚不明晰。[目的] 利用马利亚霉菌（Mariannaea sp.） HJ合成了3种金属硒化物并对其合成机理进行了初步探索。[方法] 利用X射线衍射（X-Ray Diffraction,XRD）和傅里叶转换红外线光谱（Fourier Transform Infrared Spectroscopy,FTIR）对菌株HJ合成的金属硒化物进行了初步的表征,考察了纳米材料合成过程中总巯基含量、总抗氧化性能及自由基含量变化,并且验证了转运蛋白DMT1在金属硒化物合成中所起的关键性作用。[结果] XRD结果表明菌株HJ能够在Bi³⁺、Pb²⁺、Co²⁺与SeO₃^2-作用下分别合成Bi₄Se₃、PbSe和CoSe₂纳米颗粒,其合成的最优pH条件分别为6.0、7.0、8.0。FTIR结果表明,合成的金属硒化物表面含有氨基、羧基、羟基等官能团。3种金属硒化物的合成反应体系与空白对照组相比,总巯基含量明显下降,而总抗氧化性能却有所提高,这表明巯基等酶促体系或氨基酸金属蛋白类的非酶促体系可能参与了SeO₃^2-的还原过程。苄基异硫脲盐酸盐屏蔽实验表明,转运蛋白DMT1在SeO₃^2-转运和金属硒化物分泌过程中起到关键作用。此外,Bi³⁺、Pb²⁺和Co²⁺的加入使得菌株HJ产生氧化应激反应,在胞外分泌了大量的过氧化氢、羟基自由基和超氧自由基,而上述自由基可通过诱导热激效应的方式增强金属离子或纳米颗粒的转运过程。[结论] 利用马利亚霉菌（Mariannaeasp.） HJ合成了Bi₄Se₃、PbSe和CoSe₂纳米颗粒,为研究金属硒化物的生物合成及机理提供了一定的理论参考。     

Synthesis of copper/nickel nanoparticles using newly synthesized Schiff-base metals complexes and their cytotoxicity/catalytic activities

Transition metal complexes compounds with Schiff bases ligand representing an important class of compounds that could be used to develop new metal-based anticancer agents and as precursors of metal NPs. Herein, 2,3-bis-[(3-ethoxy-2-hydroxybenzylidene)amino]but-2-enedinitrile Schiff base ligand and its corresponding copper/nickel complexes were synthesized. Also, we reported a facile and rapid method for synthesis nickel/copper nanoparticles based on thermal reduction of their complexes. Free ligand, its metal complexes and metals nanoparticles have been characterized based on elemental analysis, transmission electron microscopy, powder X-ray diffraction, magnetic measurements and by various spectroscopic (UV–vis, FT-IR, ¹H NMR, GC–MS) techniques. Additionally, the in vitro cytotoxic activity of free ligand and its complexes compounds were assessed against two cancer cell lines (HeLa and MCF-7 cells)and one healthy cell line (HEK293 cell). The copper complex was found to be active against these cancer cell lines at very low LD₅₀ than the free ligand, while nickel complex did not show any anticancer activity against these cell lines. Also, the antibacterial activity of as-prepared copper nanoparticles were screened against Escherichia coli, which demonstrated minimum inhibitory concentration and minimum bactericidal concentration values lower than those values of the commercial Cu NPs as well as the previous reported values. Moreover, the synthesized nickel nanoparticles demonstrated remarkable catalytic performance toward hydrogenation of nitrobenzene that producing clean aniline with high selectivity (98%). This reactivity could be attributed to the high degree of dispersion of Ni nanoparticles.     

CONVERSION OF TRYPSIN TO A COPPER ENZYME: TYROSINASE/CATECHOL OXIDASE BY CHEMICAL MODIFICATION

New active sites can be introduced into naturally occurring enzymes by the chemical modification of specific amino acid residues in concert with genetic techniques. Chemical strategies have had a significant impact in the field of enzyme design such as modifying the selectivity and catalytic activity which is very different from those of the corresponding native enzymes. Thus, chemical modification has been exploited for the incorporation of active site binding analogs onto protein templates and for atom replacement in order to generate new functionality such as the conversion of a hydrolase into a peroxidase. The introduction of a coordination complex into a substrate binding pocket of trypsin could probably also be extended to various enzymes of significant therapeutic and biotechnological importance.

The aim of this study is the conversion of trypsin into a copper enzyme: tyrosinase by chemical modification. Tyrosinase is a biocatalyst (EC.1.14.18.1) containing two atoms of copper per active site with monooxygenase activity. The active site of trypsin (EC 3.4.21.4), a serine protease was chemically modified by copper (Cu⁺²) introduced p-aminobenzamidine (pABA- Cu⁺²: guanidine containing schiff base metal chelate) which exhibits affinity for the carboxylate group in the active site as trypsin-like inhibitor. Trypsin and the resultant semisynthetic enzyme preparation was analysed by means of its trypsin and catechol oxidase/tyrosinase activity. After chemical modification, trypsin-pABA-Cu⁺² preparation lost 63% of its trypsin activity and gained tyrosinase/catechol oxidase activity. The kinetic properties (K_cat, K_m, K_cat/K_m), optimum pH and temperature of the trypsin-pABA-Cu⁺² complex was also investigated.     

Conjugation of nattokinase and lumbrukinase with magnetic nanoparticles for the assay of their thrombolytic activities

Two important thrombolytic enzymes, nattokinase (NK) and lumbrukinase (LK), were immobilized onto fine magnetic Fe₃O₄ nanoparticles using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) as the coupling reagent, and their thrombolytic activities were studied. The Fe₃O₄ nanoparticles and NK- and LK-conjugated magnetic nanoparticles were characterized by transmission electron microscopy, Fourier transform infrared spectrophotometry, vibrating sample magnetometry, X-ray diffraction, and UV–vis absorption spectroscopy. Dual kinetic absorbance measurements at 405 and 630 nm were employed to measure their thrombolytic activity. Analysis of protein amount showed that the optimum conditions for NK and LK binding to nanoparticles were respectively at a mass ratio of 2:1:1, 2:1:2 (magnetic nanoparticles:protein:EDC), and pH 6.00. Thrombolytic activity assay showed that the best thrombolytic activity could reach 91.89% for NK–nanoparticle conjugates and 207.74% for LK–nanoparticle conjugates, which are much higher than the pure enzymes (NK, 82.86%; LK, 106.57%).     

Does the peroxidase-like activity of sodium dodecyl sulphate-modified cytochrome c increase after peroxynitrite or radiation treatment?

The peroxidase-like activity of cytochrome c is considerably increased by unfolding of the protein. The enhancement of the activity is due to the higher reaction rate of unfolded cytochrome c with hydrogen peroxide, which is the rate-determining step in the peroxidase cycle of cytochrome c (Gebicka, L., 2001, Res Chem Intermed 27, 717-23). In this study we checked whether combined action of two unfolding factors, SDS and peroxynitrite or radiation (hydroxyl radicals), increases the peroxidase-like activity of cytochrome c more than any single treatment alone. Peroxynitrite reacts with SDS-modified cytochrome c in the same way as with native cytochrome c, via intermediate radical products, *OH/*NO2, arising from peroxynitrite homolysis. We found that SDS-modified cytochrome c is much more sensitive to oxidative damage than the native protein. Partial unfolding of cytochrome c by SDS causes the peroxide substrate to have a better access to the heme center. On the other hand, the amino acids located in the vicinity of the active site and/or heme group become accessible for oxidizing radicals. The overall effect observed is that the peroxidase-like activity of SDS-modified cytochrome c decreases with an increase of the concentration of the oxidizing species (peroxynitrite or radiolytically generated hydroxyl radicals). The damage of SDS-modified cytochrome c caused by irradiation is much more significant than that observed after peroxynitrite treatment.     

Superoxide dismutase–mimicking activities of dinuclear Cu(II) complexes with ligands containing a tetrathioether-tetraamino moiety

The superoxide scavenging activities of copper(II) complexes with the ligands, 6,6′-methylene-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L), and 6,6′-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L′), were investigated by xanthine–xanthine oxidase (X/XO) assays using nitroblue tetrazolium (NBT) as indicator molecule, and the results were compared with respect to the particular type of anion (ClO·4, Cl^·, NO·3) on the apical site of the copper(II) complexes. All of the complexes inhibited the reduction of NBT by superoxide radicals, with the [Cu₂(L′)](ClO₄)₂ complex exhibiting the highest scavenging activity against superoxide radicals among the complexes examined. The catalytic efficiency of the complexes for dismutation of superoxide radicals depends on the particular anion liganded to Cu(II) ion in the complexes, and the order of potency was observed to be ClO₄ > Cl > NO·3 in phosphate buffer at pH 7.40. The Cu(II)-H₄L′ complexes had the lowest IC₅₀ and catalytic rate constant values indicating that the distorted geometry of the Cu(II)-H₄L′ complexes influence their catalytic activities for dismutation of superoxide radicals more efficiently. The difference in the activities of the complexes toward superoxide radicals can also be attributed to the nature of the anions on the apical site of the copper(II) complexes and the superoxide dismutase-like activity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 53–59, 1998     

Copper Oxide Nanoparticles Greener Synthesis Using Tea and its Antifungal Efficiency on Fusarium solani

Abstract

A great deal of research has been done on various uses of copper oxide. The synthesis of copper oxide nanoparticles was mediated using tea extract. The first sign of the reduction of copper ions to copper oxide was the change in color of extract to dark brown after treating with copper chloride. The resulting nanoparticles were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrometry (FTIR). Finally, the antimicrobial effects of these nanoparticles on Fusarium solani were studied in vitro by agar dilution method. The TEM images showed the synthesis of copper oxide with size of less than 80?nm. The synthesized copper oxide nanoparticles showed significant inhibitory effects on F. solani cultures so that the concentration of 80?μg/ml prevented approximately 90% of the mycelium growth of the fungus. The results showed that the inhibition zone of silver nanoparticles strongly depends on their concentration and increases by increasing the concentration of copper oxide nanoparticles in the medium.     

Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath)

We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.     

Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability

To obtain regenerable magnetic nanoparticles, triethoxy(3-isocyanatopropyl)silane and iminodiacetic acid (IZ) were used as the starting material and immobilized on Fe₃O₄ nanoparticles. Copper ions (Cu²⁺ ions) were loaded on the Fe-IZ nanoparticles and used for cellulase immobilization. The support was characterized by spectroscopic methods (FTIR, NMR) and thermogravimetric analysis, transmission electron microscopy, scanning electron microscope, X-ray diffraction, energy dispersive X-ray analysis, and vibrating sample magnetometer techniques. As a result of experiments, the amount of protein bound to immobilized cellulase (Fe-IZ-Cu-E) and cellulase activity was found to be 33.1 mg/g and 154 U/g at pH 5, 50°C, for 3 h. The results indicated that the free cellulase had kept only 50% of its activity after 2 h, while the Fe-IZ-Cu-E was observed to be around 77%, at 60°C. It was found that the immobilized cellulase maintained 93% of its initial catalytic activity after its sixth use. Furthermore, the Fe-IZ-Cu-E retained about 75% of its initial activity after 28 days of storage. To reuse the support material (Fe-IZ-Cu), it was regenerated by thorough washing with ammonia or imidazole.     

Cadmium and copper induction of oxidative stress and antioxidative response in tomato (<Emphasis Type="Italic">Solanum lycopersicon</Emphasis>) leaves

We compare cadmium and copper induced oxidative stress in tomato leaves and the antioxidative enzyme response during a time course of 96 h. Plants were subjected to 25 μM of CdCl₂ or CuSO₄ and malondialdehyde (MDA) level and activity of guaiacol peroxidase, superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were determined. The results showed that there was an early increase in the MDA level and in the guaiacol peroxidase activity more pronounced with copper exposure during almost all the time course of the experiment. The activity of superoxide dismutase and catalase was induced very early after cadmium and copper treatment, reached a maximal value after 12 h and then declined but it remained always slightly higher than the control at the end of the experiment. Ascorbate peroxidase activity pathway was similar to superoxide dismutase or catalase with a maximal activity after 48 h of heavy metal exposure. Induction of glutathione reductase activity observed only under copper exposure is maintained during almost all the experimental time. The antioxidative activity developed by tomato leaves is more induced by copper treatment. This can be related to the ability of this metal to induce more than cadmium an accumulation of reactive oxygen species (ROS) at the cellular level. Decline in the antioxidative enzymes activity at the end of the experiment can be a consequence of cadmium- and copper-inducing a further ROS formation that might affect enzymes activity.     

Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of cold-active protein-tyrosine phosphatase from a psychrophile, Shewanella sp

The cold-active protein-tyrosine phosphatase (CAPTPase) of a psychrophile, Shewanella sp., shows high catalytic activity below 20 degrees C. The catalytic residue of CAPTPase is histidine, as opposed to the cysteine of known protein-tyrosine phosphatases (PTPases), and the enzyme protein has three amino acid sequences, Asp-Xaa-His, Gly-Asp-Xaa-Xaa-Asp-Arg and Gly-Asn-His-Glu, that are observed in many protein-serine/threonine phosphatases (PS/TPases). We have determined the crystal structures of CAPTPase at 1.82 angstroms and the enzyme bound with a phosphate ion at 1.90 angstroms resolution using X-ray crystallography and the multiple isomorphous replacement method. The final refined models are comprised of 331 amino acid residues, two metal ions, 447 water molecules, and an acetate or phosphate ion in an asymmetric unit. The enzyme protein consists of three beta-sheets, termed Sheet I, Sheet I', and Sheet II, and 14 alpha-helices. The CAPTPase has a different overall structure from known protein-tyrosine phosphatases. The arrangement of two metal ions, a phosphate ion and the adjacent amino acid residues in the catalytic site of CAPTPase is identical to that of PS/TPases. Thus, it was confirmed that the CAPTPase was a novel PTPase with a conformation similar to the catalytic site of PS/TPase. We speculate that the hydrophobic moiety around the catalytic residue of CAPTPase might play an important role in eliciting high activity at low temperature.