Multicatalytic Activity Research Articles

‘One-pot’ engineering of 0D carbon-based inorganic nanoarchitectonics for boosting multitasking catalytic activity

Easy-to-make nanostructured materials that exhibit multitasking activity —while reducing the use of hazardous solvents throughout its architectural design— is a must to advance in the so-called Industry 4.0. Herein, a simple and eco-friendly ‘one-pot’ functionalization approach has been devised for synthesizing 0D carbon-based inorganic nanoarchitectonics made of carbon dots (CDs, as core carbon source) carrying two different inorganic building blocks, viz. quantum dots (CdSe@ZnS–QDs) and metal nanoparticles (Pt–NPs). As a proof-of-principle, the multi-catalytic activity of the resulting 0D Pt/QD/CD nanoarchitectonics by means of photoelectrocatalysis and sonocatalysis has been considered and compared with the pristine CD counterpart, demonstrating its suitability for boosting pivotal catalytic tasks like i) the photoelectrogeneration of hydrogen via hydrogen evolution reaction (HER), and ii) the sonodegradation of environmental pollutants (i.e., Rhodamine B) in water. Overall, this chemical approach is general and might be tailored for architecting alternative carbon-based heterostructures to enhance alternative multi-catalytic tasks.

Tri-functional carbon nanocages coated CoNi electrocatalyst with micro twinning structure for high-performance electrochemical devices

The twinning structure could lead to unexpected effects on electrocatalytic performance; however, research on relevant mechanisms is still rare and there is still a lack of an effective and convenient strategy to introduce twinning structures into nanoparticles. In this work, inspired by the in-situ fcc-hcp phase transition in metals and benefiting from the features of Prussian blue analogues (PBAs), N-doped carbon cages-coated CoNi nanoparticles, which contain multi-active sites including alloyed nanoparticles, micro twinning structures and single atom sites could be effectively fabricated by pyrolyzing of CoNi-based PBAs. The catalyst exhibits an obvious enhancement in oxygen evolution/reduction reaction and hydrogen evolution reaction under alkaline conditions. Theoretical calculations verify that the micro-twinning structures originating from the fcc-hcp phase transition promote electrocatalytic activities significantly due to the optimized adsorption energy towards different intermediates. Moreover, the synergic effect between the multi-active sites may further optimize the electronic structures and lead to the deviation from the Scaling Effect. The water-splitting device and Zinc-Air battery are also assembled based on this micro- twinning electrocatalyst, which yields a maximum power density of 95 mW cm−2 and a low voltage of 1.76 V to reach 10 mA cm−2 current density, respectively. In all, this work contributes a simple and universal strategy to introduce both the multi-active sites and the twinning structure into transition metal-based catalysts, which improves the multi-catalytic activity towards different reactions significantly.

Multienzyme-Like Nanozymes: Regulation, Rational Design, and Application.

Nanomaterials with more than one enzyme-like activity are termed multienzymic nanozymes, and theyhave received increasing attention in recent years and hold huge potential to be applied in diverse fields, especially for biosensing and therapeutics. Compared to single enzyme-like nanozymes, multienzymic nanozymes offer various unique advantages, including synergistic effects, cascaded reactions, and environmentally responsive selectivity. Nevertheless, along with these merits, the catalytic mechanism and rational design of multienzymic nanozymes are more complicated and elusive as compared to single-enzymic nanozymes. In this review, the multienzymic nanozymes classification scheme based on the numbers/types of activities, the internal and external factors regulating the multienzymatic activities, the rational design based on chemical, biomimetic, and computer-aided strategies, and recent progress in applications attributed to the advantages of multicatalytic activities are systematically discussed. Finally, current challenges and future perspectives regarding the development and application of multienzymatic nanozymes are suggested. This review aims to deepen the understanding and inspire the research in multienzymic nanozymes to a greater extent.

Functional molecule-mediated assembled copper nanozymes for diabetic wound healing

BackgroundThe complex hyperglycemic, hypoxic, and reactive oxygen species microenvironment of diabetic wound leads to vascular defects and bacterial growth and current treatment options are relatively limited by their poor efficacy.ResultsHerein, a functional molecule-mediated copper ions co-assembled strategy was constructed for collaborative treatment of diabetic wounds. Firstly, a functional small molecule 2,5-dimercaptoterephthalic acid (DCA) which has symmetrical carboxyl and sulfhydryl structure, was selected for the first time to assisted co-assembly of copper ions to produce multifunctional nanozymes (Cu-DCA NZs). Secondly, the Cu-DCA NZs have excellent multicatalytic activity, and photothermal response under 808 nm irradiation. In vitro and in vivo experiments showed that it not only could efficiently inhibit bacterial growth though photothermal therapy, but also could catalyze the conversion of intracellular hydrogen peroxide to oxygen which relieves wound hypoxia and improving inflammatory accumulation. More importantly, the slow release of copper ions could accelerate cellular proliferation, migration and angiogenesis, synergistically promote the healing of diabetic wound furtherly.ConclusionsThe above results indicate that this multifunctional nanozymes Cu-DCA NZs may be a potential nanotherapeutic strategy for diabetic wound healing.

Triggering electrode multi-catalytic activity for reversible symmetric solid oxide cells by Pt-doping lanthanum strontium ferrite

Among the up-and-coming technologies, reversible solid oxide cells (r-SOCs) represent a highly efficient way to store and convert energy using a single device. The interchangeability between SOFC and SOEC operation is the key aspect, though for its technological advancement the development of performing, versatile and robust electrodes must be pursued. A multi-purpose catalyst would allow for the design of a reversible symmetric cell (r-SSOC), greatly reducing fabrication and maintenance costs. However, such a flexible electrode material must comply with the requirements of both SOC electrodes, namely ensuring high performance in a wide pO2 range: catalytic activity towards fuel oxidation and H2O/CO2 reduction, electronic and ionic conductivity, long-term stability, tolerance to carbon deposition. In this work, platinum doping at the B-site of a lanthanum strontium ferrite (LSFPt) endowed the parent perovskite with superior versatility, making it able to operate as oxygen exchange catalyst as well as fuel electrode material. 5-mol% Pt inclusion enhanced the ORR/OER activity, lowering the LSF area-specific resistance by 29% and approaching the state-of-the-art LSCF performance. After reduction, Pt-Fe finely distributed exsolution provided the fuel electrode with catalytically active sites. LSFPt-symmetric H2-SOFCs and CO2-SOECs, displayed 720 mW∙cm−2 power density and 1.66 A·cm−2 current density at 1.6 V, respectively, at 850 °C. Endurance tests on LSFPt-based r-SSOCs demonstrated a remarkably stable SOFC/SOEC cyclability and a steady current density output for over 370 h in 50:50 CO2:CO at 850 °C.

Functional characterization of secologanin synthase-like homologous genes suggests their involvement in the biosynthesis of diverse metabolites in the secoiridoid biosynthetic pathway of Camptotheca acuminata Decne

The assignment of functions based on homology has recently been challenged by the frequent discovery of functional divergence among homologous gene family members of enzymes involved in plant secondary metabolism. Secologanin synthase (SLS) is the key CYP450 enzyme that acts critically in the biosynthesis of Strychnos alkaloid scaffold. In this study, to fully elucidate the mechanism that underlies metabolic variation, the CYP450 paralogs that participate in oxidative transformation of the secoiridoid pathway were functionally characterized by combining multitiered strategies of metabolite profiling, phylogenetic analyses, biochemistry assays and reverse genetics techniques. Five CaSLSs-like homologous genes were mined and isolated from an integrative multi-omics database of Camptotheca acuminata. Protein sequences, structural comparisons, and phylogenetic analyses confirmed that CaSLS1–2 and CaSLS4–5 were grouped into the SLS clade, and only CaSLS3 clustered into the 7DLH clade. Five homologs, including two previously identified enzymatic genes, were thus designated as CaSLAS1, CaSLAS2, Ca7DLH, CaSLS4 and CaSLS5. Enzymatic assays of the recombinant proteins in yeast showed that CaSLAS1 and CaSLAS2 displayed multi-catalytic activities of SLS, secologanic acid synthase (SLAS) and secoxyloganin synthase (SXS). Additionally, the reactions of CaSLASs enzymes generated stereospecific isomers of secoiridoid products, and a new product of secoxyloganin was observed. CaSLS5, a third SLS enzyme isoform that catalyzes the formation of secologanin, was reported for the first time. However, CaSXS enzymatic activities in vitro had little physiological impact on the biosynthesis of camptothecin (CPT) in Camptotheca acuminata. The primary and secondary roles of CaSLSs-like genes in secoiridoid metabolism were confirmed by virus-induced gene silencing (VIGS) in plant leaves. Efficient silencing and transcriptional downregulation of CaSLAS2, compared with the CaSLAS1 homologs, resulted in a greater reduction of the accumulation of CPT within silenced plants, and CaSLS5 had barely any effect on the contents of metabolites in planta. Thus, CaSLAS2, rather than CaSLAS1, appeared to function as a major participant in the biosynthesis of CPT, and there were redundant functions in the CaSLSs-like enzymes. Consistent with such roles, CaSLAS2 was ubiquitously expressed at very high levels in Camptotheca tissues, and CaSLAS2 was specifically expressed in young leaves. In contrast, CaSLS5 was poorly expressed in every tissue tested. Our findings demonstrate that homologs that belong to the CYP72 gene family are functionally diverse and exhibit divergence and thereby uncover an expanding group of enzymatic genes that determine the chemo-diversity of the iridoid pathway.

Cu-Doped Polypyrrole with Multi-Catalytic Activities for Sono-Enhanced Nanocatalytic Tumor Therapy.

Nanocatalytic medicine is a burgeoning disease treatment model with high specificity and biosafety in which the nanocatalyst is the core of driving catalytic reaction to generate therapeutic outcomes. However, the robust defense systems in the pathological region would counteract nanocatalyst-initiated therapeutics. Here, a Cu-doped polypyrrole is innovatively developed by a facile oxidative polymerization reaction, which exhibits intriguing multi-catalytic activities, including catalyzing H2 O2 to generate O2 and · OH, and consuming reduced glutathione by a Cu(II)-Cu(I) transition approach. By decorating with sonosensitizers and DSPE-PEG, the obtained CuPPy-TP plus US irradiation can induce severe oxidative damage to tumor cells by amplifying oxidative stress and simultaneously relieving antioxidant capacity in tumors based on the highly effective sonochemical and redox reactions. The notable tumor-specific biodegradability, remarkable cell apoptosis in vitro, and tumor suppression in vivo are demonstrated in this work, which not only present a promising biocompatible antitumor nanocatalyst but also broaden the perspective in oxidative stress-based antitumor therapy.

Prussian Blue: A Nanozyme with Versatile Catalytic Properties.

Nanozymes, nanomaterials with enzyme-like activities, are becoming powerful competitors and potential substitutes for natural enzymes because of their excellent performance. Nanozymes offer better structural stability over their respective natural enzymes. In consequence, nanozymes exhibit promising applications in different fields such as the biomedical sector (in vivo diagnostics/and therapeutics) and the environmental sector (detection and remediation of inorganic and organic pollutants). Prussian blue nanoparticles and their analogues are metal–organic frameworks (MOF) composed of alternating ferric and ferrous irons coordinated with cyanides. Such nanoparticles benefit from excellent biocompatibility and biosafety. Besides other important properties, such as a highly porous structure, Prussian blue nanoparticles show catalytic activities due to the iron atom that acts as metal sites for the catalysis. The different states of oxidation are responsible for the multicatalytic activities of such nanoparticles, namely peroxidase-like, catalase-like, and superoxide dismutase-like activities. Depending on the catalytic performance, these nanoparticles can generate or scavenge reactive oxygen species (ROS).

An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 μA cm-2 mM-1 and a LOD of 0.4 μM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Dye degradation and bacterial disinfection using multicatalytic BaZr0.02Ti0.98O3 ceramics

AbstractThe piezocatalytic, photocatalytic, and piezo‐photocatalytic properties of BaZr0.02Ti0.98O3 (BZT) were examined for degradation of organic dyes and killing of bacteria present in the wastewater. Poled and unpoled powder samples were investigated using rhodamine‐B (RB), methyl orange (MO) dyes, and gram‐negative Escherichia coli in simulated water. The poled BZT sample demonstrated significant degradation of RB and MO dyes as compared to the unpoled BZT sample during piezocatalytic, photocatalytic, and piezo‐photocatalytic experiments. Moreover, ultrasonication‐induced piezocatalysis was found to be more effective than stirring in dye degradation using the poled BZT sample. Also, the piezo‐photocatalysis through poled BZT sample (using ultrasonication and ultraviolet (UV) light) was found to be more effective in dye degradation than that of only piezocatalysis and only photocatalysis. During antibacterial testing, the poled BZT sample showed nearly 90% and 100% bacterial killing under UV light and ultrasonication, respectively,, whereas the unpoled BZT sample showed only ~5% and ~35% bacterial killing. The significant enhancement in dye degradation and bacterial killing using the poled sample explicitly indicated the role of internal electric field in multicatalytic activities.

Multicatalytic behavior of Ba0.85Ca0.15Ti0.9 Zr0.1O3 ceramics for pharmaceutical/dye/bacterial treatments

Multicatalytic activities (photocatalysis, piezocatalysis, and pyrocatalysis) of ferroelectric ceramics Ba0.85Ca0.15Ti0.9Zr0.1O3 (BCZTO) were studied. Maximum degradations (89% and 81%) were achieved in piezo-photocatalytic experiments for degradation of Rhodamine B (RB) and ciprofloxacin. Similarly, 95% degradation of RB was achieved during pyrocatalysis in 250 heating/cooling cycles. Antibacterial performance of ceramics was analyzed with the help of Gram-positive and Gram-negative bacteria-killing processes. The bacterial colony formation drops to zero in 90 min with poled samples of BCZTO ceramics. The poled samples performed much better than that of the unpoled samples in all the catalytic reactions as well as in the bacterial killing process.

3D-printed CuO nanoparticle-functionalized flow reactor enables online fluorometric monitoring of glucose.

An online flow reactor was fabricated by using a fused deposition modeling three-dimensional printing (3DP) technology along with thermoplastic poly(lactic acid) filaments incorporating copper oxide nanoparticles (CuO NPs). In the presence of glucose, the flow reactor displays multi-catalytic activities because accelerates the oxidation of 2',7'-dichlorodihydrofluorescein to form fluorescein which displays green fluorescence under 480nm excitation (emission wavelength: 530nm). The CuO NPs exert two functions to mediate electron transfer at a basic reaction condition, viz. direct oxidation of glucose to generate reactive oxygen species (ROS), and prompting the ROS to oxidize 2',7'-dichlorofluorescin diacetate. The flow reactor coupled to a microdialysis sampler and a fluorometer was applied for online fluorometric monitoring of brain extracellular glucose levels in living rats based on scanning of time-resolved fluorescence intensities. After optimization of (a) the manufacture of the flow reactor, (b) the reaction conditions (pH10; 50°C), and (c) the online analytical system, the detection limit of the method (when using 10-μL samples of microdialysate) is as low as 6.1μM (linear range: 0.05-5mM) with a sampling frequency of 7.5h-1. To illustrate the method's applicability, analyses of spiked off-line-collected rat brain microdialysates were conducted. In addition, rat brain extracellular glucose levels were monitored in-vivo and online upon neuronal depolarization triggered by perfusing a high-K+ medium. The results demonstrate that functionalizing raw 3DP materials with appropriate nanomaterials can simplify the manufacturing of analytical devices and related analytical procedures. This will extend the diversity and adaptability of current 3DP-enabling analytical strategies. Graphical abstract Schematic presentation of an online flow reactor fabricated using a fused deposition modeling 3D printer along with poly(lactic acid) (PLA) filaments incorporating CuO NPs. The manufactured flow reactor displays multi-catalytic activities and simplifies online fluorometric monitoring of living rat brain extracellular glucose.

Visible-light induced enhancement in the multi-catalytic activity of sulfated carbon dots for aerobic carbon–carbon bond formation

The development of carbonaceous materials as metal-free catalysts integrating different types of catalysis in a single system represents a significant advance in cascade/tandem organic synthesis.

Functional hydrogels with a multicatalytic activity for bioremediation: Single‐step preparation and characterization

ABSTRACTIn a single‐step free radical reaction, multicatalytic hydrogels were synthesized by covalent immobilization of Pancreatin onto a film composed of Acrylamide and Polyethylene glycol dimethacrylate750. Hydrogels were characterized by determination of their dynamic swelling ratios and each catalytic activity was extensively investigated by determination of kinetic parameters KM and Vmax. The immobilization process was found to preserve the hydrolytic properties of Pancreatin (Protease, Lipase, and Amylase catalytic activities). Catalytic efficiencies were the highest with Protease and the lowest with Amylase. Reusability values higher than 60% after 10 repeated cycles proved the applicability of the proposed material in industrial practice. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43338.

Cascade Reactions Catalyzed by Bionanostructures

Cascade reactions are an emerging technology in organic chemistry, introducing elegance and efficiency to synthetic strategies. This Review provides an overview of the novel and recent achievements in cascade processes catalyzed by bionanostructures. The examples here selected demonstrate the advances related to the application of heterogeneous nanocatalysts—nanostructures and biomolecules combined by different manner—in efficient cascade processes. Metallic nanoparticles supported on biomolecules, multienzymatic systems or bionanohybrid structures with multicatalytic activities (containing both organometallic and biocatalytic activity) were selectively and efficiently used alone or in cooperative fashion. This Review highlights examples of efficient and interesting catalytic cascade processes in organic chemistry, ultrasensitive biosensing, or energy storage and conversion, underscoring their tremendous future potential in chemical synthesis.

Rapid Equilibrium Kinetic Analysis of Arsenite Methylation Catalyzed by Recombinant Human Arsenic (+3 Oxidation State) Methyltransferase (hAS3MT)

In the human body, arsenic is metabolized by methylation. Understanding this process is important and provides insight into the relationship between arsenic and its related diseases. We used the rapid equilibrium kinetic model to study the reaction sequence of arsenite methylation. The results suggest that the mechanism for arsenite methylation is a completely ordered mechanism that is also of general interest in reaction systems with different reductants, such as tris(2-carboxyethyl)phosphine, cysteine, and glutathione. In the reaction, cysteine residues of recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) coordinate with arsenicals and involve the methyl transfer step. S-Adenosyl-l-methionine (AdoMet) is the first-order reactant, which modulates the conformation of hAS3MT to a best matched state by hydrophobic interaction. As the second-order reactant, reductant reduces the disulfide bond, most likely between Cys-250 and another cysteine residue of hAS3MT, and exposes the active site cysteine residues for binding trivalent inorganic arsenic (iAs(3+)) to give monomethylarsonic dicysteine (MADC(3+)). In addition, the reaction can be extended to further methylate MADC(3+) to dimethylarsinic cysteine (DAMC(3+)). In the methylation reaction, the β-pleated sheet content of hAS3MT is increased, and the hydrophobicity of the microenvironment around the active sites is decreased. Similarly, we confirm that both the high β-pleated sheet content of hAS3MT and the high dissociation ability of the enzyme-AdoMet-reductant improve the yield of dimethylated arsenicals.

Cu(ii)- and disulfide bonds-induced stabilization during the guanidine hydrochloride- and thermal-induced denaturation of NAD-glycohydrolase from the venom of Agkistrodon acutus

NAD-glycohydrolase (AA-NADase) from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. Among all identified NADases, only AA-NADase is a disulfide-linked dimer and contains Cu(2+). Cu(2+) and disulfide bonds are essential for its multicatalytic activity. In this study, the effects of Cu(2+) and disulfide-bonds on guanidine hydrochloride (GdnHCl)- and thermal-induced unfolding of AA-NADase have been investigated by fluorescence, circular dichroism (CD) and differential scanning calorimetry (DSC). Cu(2+) and disulfide bonds not only increase the free energy change during the GdnHCl-induced unfolding as determined by fluorescence, but also increase the overall enthalpy change and the transition temperature during the thermal-induced unfolding as determined by CD and DSC. The slope of the GdnHCl-induced unfolding curve at its midpoint and the heat capacity of thermal-induced unfolding are slightly affected by Cu(2+) but significantly decrease after reduction of three disulfide-bonds. This work suggests that Cu(2+) stabilizes the folded state by increasing the enthalpy of unfolding, while disulfide-bonds stabilize the folded state by increasing the enthalpy of unfolding and stabilizing the packing of hydrophobic residues. Thus both Cu(2+) and disulfide bonds play a structural role in its multicatalytic activity.

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: Regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.

Small-molecule reductants inhibit multicatalytic activity of AA-NADase fromAgkistrodon acutusvenom by reducing the disulfide-bonds and Cu(II) of enzyme

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(II) and has disulfide-bond linkages between two peptide chains. The effects of the reduction of the disulfide-bonds and Cu(II) in AA-NADase by small-molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA-NADase has six disulfide-bonds and fifteen free cysteine residues. L-ascorbate inhibits AA-NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA-NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2-carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide-bonds in AA-NADase. Apo-AA-NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo-AA-NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide-bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA-NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L-ascorbate are endogenous inhibitors to the multicatalytic activity of AA-NADase.

Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles.

The cysteine protease known as "prohormone thiol protease" (PTP) has been identified as a major proenkephalin processing enzyme in secretory vesicles of adrenal medulla (known as chromaffin granules). This study provides the first demonstration that PTP exists as a multicatalytic cysteine protease complex that can be activated by endogenous glutathione present in chromaffin granules. The high molecular mass nature of PTP, of approximately 185 kDa, was demonstrated by elution of a single peak of 35S-enkephalin precursor cleaving activity by Sephacryl S200 gel filtration chromatography and by a single band of 35S-enkephalin precursor cleaving activity detected on radiozymogram gels under native buffer conditions. Importantly, when 0.1% SDS was included in radiozymogram gels, PTP activity was resolved into three bands of proteolytic activity with apparent molecular masses of 88, 81, and 61 kDa. These activities were all cysteine proteases, since they were inhibited by the cysteine protease inhibitor E-64c but not by pepstatin A or EDTA that inhibit aspartyl protease and metalloprotease, respectively. Purification of native PTP by preparative gel electrophoresis indicated that PTP was composed of four polypeptides of 66, 60, 33, and 29 kDa detected on SDS-PAGE gels. These four protein subunits accounted for the three catalytic activities of PTP, as demonstrated on 35S-enkephalin precursor radiozymogram gels. Results also indicated that the electrophoretic mobilities of the four subunits differed under reducing compared to nonreducing conditions. The multicatalytic activities of the PTP complex all require reducing conditions for activity, which can be provided by endogenous reduced glutathione in chromaffin granules. These novel findings provide the first evidence for a role of a multicatalytic cysteine protease complex, PTP, in chromaffin granules that may be involved in the proteolytic processing of proenkephalin and perhaps other precursors into active neuropeptides.