Presence Of Divalent Metal Ions Research Articles

Metal Ion-Induced Formation of Metallogels by Visible-Light-Responsive Phenylalanine-Functionalized Arylazopyrazole Ligands

A visible-light-responsive arylazopyrazole-functionalized phenylalanine (4-MeS-AAP-NF) derived ligand was designed and synthesized, and it was found to form metallogels with reversible photo-responsive properties in mixed methanol/water (MeOH/H2O) solvents. The gelation behavior of the 4-MeS-AAP-NF ligand in the presence of different divalent metal ions in mixed methanol/water (MeOH/H2O) solvents at pH~11.60 was studied. It was found that the 4-MeS-AAP-NF ligand alone could not self-assemble to form any gels. However, in the presence of divalent metal ions, it readily formed the assembled metallogels in an alkaline aqueous/methanol solution with various morphologies. The results suggest that the gelation process was triggered by divalent metal ions. The presence of the AAP moiety in the gel matrix rendered the metallogel assemblies photo-responsive, and the reversible gel-to-sol phase transition was studied by UV-vis spectroscopy. The gels showed a slow, reversible visible-light-induced gel-to-sol phase transition under blue (λ = 405 nm) and then sol-to-gel transition by green light (λ = 530 nm) irradiation, resulting in the re-formation of the original gel state. The morphology and viscoelastic properties of the yellow–orange opaque metallogels were characterized by scanning electron microscopy (SEM) and rheological measurement, respectively.

Plant Organellar MSH1 Is a Displacement Loop-Specific Endonuclease.

MutS HOMOLOG 1 (MSH1) is an organellar-targeted protein that obstructs ectopic recombination and the accumulation of mutations in plant organellar genomes. MSH1 also modulates the epigenetic status of nuclear DNA, and its absence induces a variety of phenotypic responses. MSH1 is a member of the MutS family of DNA mismatch repair proteins but harbors an additional GIY-YIG nuclease domain that distinguishes it from the rest of this family. How MSH1 hampers recombination and promotes fidelity in organellar DNA inheritance is unknown. Here, we elucidate its enzymatic activities by recombinantly expressing and purifying full-length MSH1 from Arabidopsis thaliana (AtMSH1). AtMSH1 is a metalloenzyme that shows a strong binding affinity for displacement loops (D-loops). The DNA-binding abilities of AtMSH1 reside in its MutS domain and not in its GIY-YIG domain, which is the ancillary nickase of AtMSH1. In the presence of divalent metal ions, AtMSH1 selectively executes multiple incisions at D-loops, but not other DNA structures including Holliday junctions or dsDNA, regardless of the presence or absence of mismatches. The selectivity of AtMSH1 to dismantle D-loops supports the role of this enzyme in preventing recombination between short repeats. Our results suggest that plant organelles have evolved novel DNA repair routes centered around the anti-recombinogenic activity of MSH1.

Riemerella anatipestifer Endonuclease I displays enzymatic activity and is associated with bacterial virulence

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. We previously reported that the R. anatipestifer AS87_RS02625 is a secretory protein of the type IX secretion system (T9SS). In this study, R. anatipestifer T9SS protein AS87_RS02625 was determined to be a functional Endonuclease I (EndoI), which has DNase and RNase activities. Optimal temperature and pH of the recombinant R. anatipestifer EndoI (rEndoI) to cleave λDNA were determined as 55–60 °C and 7.5 respectively. The DNase activity of the rEndoI was dependent on the presence of divalent metal ions. Presence of Mg2+ at a concentration range of 7.5–15 mM in the rEndoI reaction buffer displayed the highest DNase activity. In addition, the rEndoI displayed RNase activity to cleave MS2-RNA (ssRNA), either in the absence or presence of divalent cations Mg2+, Mn2+, Ca2+, Zn2+ and Cu2+. The DNase activity of the rEndoI was significantly enhanced by Mg2+, Mn2+ and Ca2+ but not Zn2+ and Cu2+. Moreover, we indicated that R. anatipestifer EndoI functioned on the bacterial adherence, invasion, in vivo survival and inducing inflammatory cytokines. These results indicate that the R. anatipestifer T9SS protein AS87_RS02625 is a novel EndoI, displays endonuclease activity and plays an important role in bacterial virulence.

Specific DNAzymes cleave the 300-618 nt of 5'UTR to inhibit DHAV-1 translation and replication.

DNAzymes effectively inhibit the expression of viral genes. Duck hepatitis A virus type-1 (DHAV-1) genomic RNA carries an internal ribosome entry site (IRES). The IRES initiates the translation of DHAV-1 via a mechanism that differs from that of cap-dependent translation. Therefore, it is an attractive target for the treatment of DHAV-1. In this study, we designed 6 DNAzymes (Dzs) specifically targeting 300-618 nt sequence in the DHAV-1 5'untranslated region (UTR; a predicted IRES-like element). In the presence of divalent metal ions, three designed DNAzymes (DZ369, DZ454, and DZ514) efficiently cleaved the 300-618 nt sequence of the DHAV-1 5'UTR RNA. The activity of the Dzs was particularly dependent on Mg2+ ions. Subsequently, the translation inhibitory activity of these Dzs was determined by western blotting experiments. The Dzs effectively inhibited the translation mediated by the 300-618 nt of DHAV-1 5'UTR in duck embryo fibroblasts (DEFs). Importantly, DZ454 showed the strongest inhibitory effect, and its inhibition was time and dose dependent. However, none of the Dzs showed significant inhibition of cap-dependent translation. These results suggest that these Dzs show specificity for target RNA. Moreover, DZ454 inhibited the replication of DHAV-1. In conclusion, the designed DNAzymes can be used as inhibitors of DHAV-1 RNA translation and replication, providing new insights useful for the development of anti-DHAV-1 drugs.

Influence of monovalent and divalent cations on monazite flotation

Understanding of flotation mechanism of monazite with oleate collector in saline water is essential for the rare earth mining industry. This work investigates the flotation performance of monazite in the presence of different ions such as Ca2+, Mg2+, Na+, and K+. The research integrates flotation experiments with colloid and surface characterizations using zeta potential, XPS and UV adsorption. The results showed that the presence of divalent metal ions (Mg2+, Ca2+) was highly detrimental to monazite flotation while the presence of monovalent metal ions (K+ and Na+) affected monazite flotation slightly only. The reason is that oleate formed insoluble products with divalent metal ions while the opposite is true in the case of monovalent metal ions. Therefore, the amount of soluble oleate ions in the presence of divalent ions was significantly reduced, resulting in less adsorption of oleate ions on monazite particles and thus reduced monazite recovery. The reduced adsorption of oleate on monazite was confirmed using XPS and UV adsorption experiments. This work explains the detrimental effect of various metal ions in saline water on monazite flotation.

Thermoresponsiveness of Carboxylated Polyallylamines Induced by Divalent Counterions as Ionic Effectors

We describe novel ionic effector-induced lower critical solution temperature (LCST)-type thermoresponsive polymers in an aqueous system. Carboxylated polyallylamines (carboxylated PAAs) are conjugates between polyallylamine and various carboxylic anhydrides possessing six-membered rings such as phthalic anhydride and 1-cyclohexene-1,2-dicarboxylic anhydride. These polymers showed LCST-type thermoresponsiveness in the presence of divalent metal ions (Ca2+, Mg2+, or Sr2+) in aqueous media, where a reversible phase transition between soluble and insoluble states was observed with a change in the temperature, although these polymers did not show thermoresponsiveness without divalent ions or with monovalent ions (Li+, Na+, or K+). The cloud points of the carboxylated PAAs could be precisely controlled by not only salt concentration, pH, salt species, and side-chain structures but also the mixing ratio of different salt species.

Silver nanoparticles based spectroscopic sensing of eight metal ions in aqueous solutions

Anthropogenic releases from different outlets of industry, municipal sewage and the road traffic can give rise to higher concentrations of heavy metals in food commodities which imposes a threat to human health and environment. A simple silver nanoparticle (Ag NPs) used for the sensing of heavy metal ions, Cd2+, Cu2+, Fe2+, Hg2+, Mn2+, Ni2+, Pb2+ and Zn2+ in aqueous solution is described qualitatively and quantitatively using spectroscopic tool. FE-SEM and TEM images confirmed that the particles are spherical in shape with an average diameter of 23.4 nm. Presence of heavy metal ions with Ag NPs gives, new peak at around 925, 898, 643, 665, 688, and 838 nm for Cd2+, Cu2+, Fe2+, Hg2+, Mn2+ and Zn2+ in addition to the peak found at 410 nm for Ag NPs. Further, the addition of Ni2+ and Pb2+ metal ion solution with Ag NPs increased the SPR band from 410 nm to 436 and 462 nm respectively. Citrate functionalized Ag NPs aggregate in solution in the presence of divalent metal ions by ions-template chelating process and are easily measurable changes in the UV–vis absorption spectrum of the particles. Further, studies also confirmed the interaction of Ag NPs with metal ions using FT-IR spectroscopy. The proposed method was found to be useful for simple UV–vis spectroscopic sensing of metal ions in aqueous solutions and real contaminated samples.

Physicochemical factors affecting flocculating properties of the proteoglycan isolated from Rhodococcus opacus

The water-soluble fraction of proteoglycan RS-89 isolated from the Rhodococcus opacus FCL89 and composed of 64.6% polysaccharide and 9.44% protein has been studied as regards its flocculating activity. The RS-89 polysaccharide component includes mannose, galactose and glucose at the molar ratio of 2.7: 1.3: 1. The basic factors affecting flocculating activity of the RS-89 have been established. Additionally, the kinetics of kaolin sedimentation without and with the bioflocculant was investigated. The presence of divalent metal ions had a positive effect on the flocculating activity of the RS-89. The addition of Ca2+ increased the RS-89 flocculating activity in comparison to the other studied metals. It was proved that the proteoglycan RS-89 achieved the highest flocculating activity at the concentration equal to 2 mg/L and in the presence of 10 mmol/L of Ca2+. The zeta potential values are less negative when there is an interaction between the kaolin particles and metal ions without the RS-89 in the tested systems. Therefore, the proposed mechanism to describe the proteoglycan interaction with kaolin particles in the presence of divalent ions includes charge neutralization and a bridging mechanism.

Solid-State NMR Study to Probe the Effects of Divalent Metal Ions (Ca2+ and Mg2+) on the Magnetic Alignment of Polymer-Based Lipid Nanodiscs.

Divalent cations, especially Ca2+ and Mg2+, play a vital role in the function of biomolecules and making them important to be constituents in samples for in vitro biophysical and biochemical characterizations. Although lipid nanodiscs are becoming valuable tools for structural biology studies on membrane proteins and for drug delivery, most types of nanodiscs used in these studies are unstable in the presence of divalent metal ions. To avoid the interaction of divalent metal ions with the belt of the nanodiscs, synthetic polymers have been designed and demonstrated to form stable lipid nanodiscs under such unstable conditions. Such polymer-based nanodiscs have been shown to provide an ideal platform for structural studies using both solid-state and solution NMR spectroscopies because of the near-native cell-membrane environment they provide and the unique magnetic-alignment behavior of large-size nanodiscs. In this study, we report an investigation probing the effects of Ca2+ and Mg2+ ions on the formation of polymer-based lipid nanodiscs and the magnetic-alignment properties using a synthetic polymer, styrene maleimide quaternary ammonium (SMA-QA), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. Phosphorus-31 NMR experiments were used to evaluate the stability of the magnetic-alignment behavior of the nanodiscs for varying concentrations of Ca2+ or Mg2+ at different temperatures. It is remarkable that the interaction of divalent cations with lipid headgroups promotes the stacking up of nanodiscs that results in the enhanced magnetic alignment of nanodiscs. Interestingly, the reported results show that both the temperature and the concentration of divalent metal ions can be optimized to achieve the optimal alignment of nanodiscs in the presence of an applied magnetic field. We expect the reported results to be useful in the design of nanodisc-based nanoparticles for various applications in addition to atomic-resolution structural and dynamics studies using NMR and other biophysical techniques.

Poly(acrylic acid) Hydrogel Microspheres for a Metal-Ion Sensor.

Monodispersed cross-linked poly(acrylic acid) (PAA) droplets (PAA X-droplets), prepared using the microfluidic method with in situ ultraviolet curing, were used as small spherical sensors to simultaneously detect both Ca2+ and Mg2+ in human saliva and serum. The PAA X-droplet treated with KOH (PAAKOH X-droplet) was used as a reference droplet because of its highly swollen state. The PAAKOH X-droplets shrunk in response to the presence of divalent metal ions (Ms) by forming a bridged structure of COO-M-OOC. The sizes of the PAAKOH X-droplets were precisely and dynamically monitored in the poly(dimethylsiloxane) (PDMS) channel with passing time when the aqueous metal-ion solutions were flowing at a controlled flow rate. The sizes of the PAAKOH X-droplets continuously decreased to the saturated constant size. The saturated size of the PAAKOH X-droplet did not change; however, the speed of size reduction increased with an increase in the concentration of the divalent metal ion. The saturated size was studied using the saturated diameter ratio (Rsat-dia) with respect to the initial diameter of the PAAKOH X-droplet before the metal-ion treatment, and the speed of the size reduction was investigated using the inverse time to reach half the saturated diameter reduction (T1/2-1). Ca2+ and Mg2+ exhibited Rsat-dia values of 75.9 and 83.6%, respectively, when the flow rate was 5 μL min-1, regardless of the metal concentration. The T1/2-1s for the Ca2+ and Mg2+ linearly increased with an increase in their concentrations. The Rsat-dia of the aqueous Ca2+/Mg2+ mixture solution had a linear relationship with φ [= CCa/(CCa + CMg), where CCa and CMg are the molar concentrations of Ca2+ and Mg2+, respectively]. The T1/2-1 of the aqueous Ca2+, Mg2+ mixture solution was calculated by adding the individual T1/2-1s of pure aqueous Ca2+ and Mg2+ solutions. Using the Rsat-dia and T1/2-1 of the Ca2+/Mg2+ mixture aqueous solution, the individual CCa and CMg in the mixture solution were successfully calculated. This method was applied to the human saliva and serum in which the major metal ions are Ca2+ and Mg2+, and other metal ions existed in undetectable amounts by the PAAKOH X-droplets. This method is simple, cost-effective, and highly accurate and solves the hurdles of separating the interference effect of a Mg2+ ion when a Ca2+ ion is measured in biofluids.

Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme–substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization–mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

Isostructural metal hydrazones of 2-(methoxycarbonyl-hydrazono)-pentanedioic acid, H2L: Synthesis, cytotoxic activity of nano spinel ZnCo2O4 derived from single source precursor, [Zn1/3Co2/3(HL)2]·3H2O

Metal Schiff base [2-(methoxycarbonyl-hydrazono)-pentanedioic acid, H2L] complexes of type [M(HL)2]·3H2O, where M = Co (1), Zn (2) & Co-Zn (3), have been synthesized by condensation of α-ketoglutaric acid and methyl carbazate in the presence of divalent metal ions. A stoichiometric reaction of the Schiff base ligand with metal ions resulted in a solid solution precursor of [Zn1/3Co2/3(HL)2]. The crystal structures of metal and mixed metal (Zn-Co) complexes were isostructural and crystallized in monoclinic systems. The synthesized solid solution was employed as a precursor for preparing zinc cobaltite spinel (ZnCo2O4) nanoparticles. As-synthesized nanospinel cobaltite was characterized using Fourier-Transform Infrared (FT-IR) spectroscopy, powder X-ray diffraction (PXRD), and transmission electron microscopy (TEM) techniques. PXRD indicated that the product had a spinel type structure with high purity and a crystalline nature. The TEM images show that the spinels were constructed via self-assembled agglomeration of nanoparticles with irregular shapes. The elemental compositions and oxidation states of as-prepared ZnCo2O4 were further analyzed by X-ray photoelectron spectroscopy (XPS). Further, the cytotoxicity was determined against human breast adenocarcinoma (MCF-7) cancer cell lines at different concentrations using a thiazolyl blue tetrazolium bromide (MTT) assay. The spinel oxides prepared from the precursors showed promising results, demonstrating their clinical importance.

Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein.

BackgroundThe Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity.Methodology/Principle findingsRecombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2’-deoxy-2’-fluoro-CTP (FdC) and 2’-deoxy-2’-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions.Conclusions/SignificanceThe requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.

Antioxidant Functions of Ergothioneine

The function of ergothioneine (EGT) has been in controversy since the discovery of EGT in 1909. The present common understanding is that the functions of EGT remain unclear and that EGT does not seem to provide any advantage over other antioxidants. In this study, the ultraviolet-absorption spectra of EGT were measured at various pH. Thermodynamical parameters such as the chemical structure and bond distances of EGT were derived by BMK/6-311+G**. The molecular orbital quantum parameters such as electron distribution, frontier electron density, highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and singly occupied molecular orbital (SOMO) were derived by HF/STO-3G. The results show that EGT exists as a resonance system mixing of thione state and ionic state in solution not only at physiological pH but also in a wide acidic pH range. EGT is very stable because of the resonance system, whereas the ionic state of EGT forms the metal complexes and the EGT-disulfide in the presence of divalent metal ions. The efficient delocalization of π-electrons on the imidazole ring of EGT increases the HOMO energy level and the rate constant of EGT towards hydroxyl (OH) radical. EGT has a high antioxidant activity by donating the highest active electron on the S atom of the imidazole ring of EGT to OH radical. Because OH radical reacts faster with EGT than any other biomolecules such as proteins and DNA, EGT consequently protects the biomolecules against oxidative damage due to the OH radical.

Dendritic Hydrogel Bioink for 3D Printing of Bacterial Microhabitat.

A glucose-modified dendritic hydrogel is used as a bioink for bacterial encapsulation. This biocompatible hydrogel is a potentially suitable alternative to conventional alginate hydrogel for bacterial encapsulation, as it readily forms gel in the presence of Na+ or K+ ions without any additional stimuli such as pH, temperature, sonication, or the presence of divalent metal ions. We created a bacterial microhabitat by adding the gelator to phosphate-buffered saline containing live bacteria at physiological pH and using an additive three-dimensional (3D) printing technique. The bacteria remained viable and metabolically active within the 3D printed bacterial microhabitat, as shown with confocal laser scanning microscopy (CLSM) and scanning electrochemical microscopy (SECM).

Magnetic Alignment of Polymer Macro‐Nanodiscs Enables Residual‐Dipolar‐Coupling‐Based High‐Resolution Structural Studies by NMR Spectroscopy

AbstractExperimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic‐resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic‐alignment behavior of lyotropic liquid‐crystalline polymer macro‐nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high‐resolution NMR. The easy preparation of macro‐nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.

Magnetic Alignment of Polymer Macro-Nanodiscs Enables Residual-Dipolar-Coupling-Based High-Resolution Structural Studies by NMR Spectroscopy.

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic-resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic-alignment behavior of lyotropic liquid-crystalline polymer macro-nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high-resolution NMR. The easy preparation of macro-nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.

Characterization of α-amylases isolated from Cyperus esculentus seeds (tigernut): Biochemical features, kinetics and thermal inactivation thermodynamics

In this study, α-amylases from fresh and dried Cyperus esculentus seeds were isolated and characterized for biochemical and thermodynamic properties. The α-amylases (FTNE-α-amylase and DTNE-α-amylase) were optimally active at 60–70 °C, pH 7.0 (FTNE-α-amylase) and pH 6.0 (DTNE-α-amylase) with activation energies (Ea) of 3.56 and 6.81 kJ mol⁻¹, respectively. Both enzymes retained above 70% of their hydrolytic activities after 1 h at pH 6.0–8.0. The enzyme activities were more enhanced in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Co²⁺, Pb²⁺, and Hg²⁺) than monovalent ions. SDS, cyclohexanol, ether and diethylamine inhibited them except EDTA. Using soluble starch as substrate, DTNE-α-amylase had higher Vₘₐₓ, Kcₐₜ and lower Kₘ than FTNE-α-amylase. Both enzymes expressed high substrate preferences for yam flour amidst other substrates with catalytic efficiencies (Kcₐₜ/Kₘ) of 2.71 and 4.44 mL. mg⁻¹sec⁻¹, respectively. The higher catalytic efficiency of DTNE-α-amylase was due to its transition state stabilization (ΔGE₋S = -0.90 kJ mol⁻¹ and ΔGE₋T = -2.22 kJ mol⁻¹). Fourier Transformed-Infrared (FTIR) spectroscopy revealed changes in C–O stretching and -N-H bending in the substrate-bound enzymes. Thermal inactivation of the amylases followed first-order rate kinetics and was non-spontaneous (ΔG#>0). FTNE-α-amylase half-lives and D-values at 70 and 80 °C, and Z-value (256.41 °C), were however higher than in DTNE-α-amylase (Z-value = 63.29 °C; t₁/₂ = 22.43 min at 70 °C) indicating that FTNE-α-amylase is more thermally stable than DTNE-α-amylase and would be more industrially applicable as a thermostable starch-hydrolyzing enzyme.

Chemical rescue of H+ delivery in proton transfer mutants of reaction center of photosynthetic bacteria

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of QB− → QBH2. In the presence of divalent metal ions (e.g. Cd2+) or in some (“proton transfer”) mutants (L210DN/M17DN or L213DN), the proton delivery to QB− is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1 M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H + Cd2+), beneath the surface (M17DN + Cd2+, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including QB− itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100–200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.

Direct Assay to Evaluate Phosphoenolpyruvate Carboxykinase Activity

Phosphoenolpyruvate carboxykinase (PEPCK) is a ubiquitous enzyme found in all known groups of organisms, acting in the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of divalent metal ion, and dependent of adenosine 5’-triphosphate (ATP) or guanosine-5’-triphosphate (GTP). PEPCK is an important enzyme in the metabolism of some organisms, such as Trypanosoma cruzi, being suggested as a potential drug target to treat Chagas’ disease. Its catalytic activity is, classically, measured by coupled assays. Herein, a direct assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying PEP, in co-elution with OAA by the differentiation obtained by the mass spectra, is reported. The developed assay was used throughout the purification protocol in order to measure the activity of PEPCK of T. cruzi, which was expressed in Escherichia coli. The purified enzyme was kinetically characterized by the developed method with Michaelis-Menten constant (KMapp) values of 96 ± 4 and 275 ± 18 µmol L-1 to OAA and ATP as substrates, respectively. The developed assay was also used for ligand screening and proved to be able to identify very low inhibitions for small molecules (50 µmol L-1).