Apparent Michaelis Constant Research Articles

Identification of positions in human aldolase a that are neutral for apparent KM

According to evolutionary theory, many naturally-occurring amino acid substitutions are expected to be neutral or near-neutral, with little effect on protein structure or function. Accordingly, most changes observed in human exomes are also expected to be neutral. As such, accurate algorithms for identifying medically-relevant changes must discriminate rare, non-neutral substitutions against a background of neutral substitutions. However, due to historical biases in biochemical experiments, the data available to train and validate prediction algorithms mostly contains non-neutral substitutions, with few examples of neutral substitutions. Thus, available training sets have the opposite composition of the desired test sets. Towards improving a dataset of these critical negative controls, we have concentrated on identifying neutral positions – those positions for which most of the possible 19 amino acid substitutions have little effect on protein structure or function. Here, we used a strategy based on multiple sequence alignments to identify putative neutral positions in human aldolase A, followed by biochemical assays for 147 aldolase substitutions. Results showed that most variants had little effect on either the apparent Michaelis constant for substrate fructose-1,6-bisphosphate or its apparent cooperativity. Thus, these data are useful for training and validating prediction algorithms. In addition, we created a database of these and other biochemically characterized aldolase variants along with aldolase sequences and characteristics derived from sequence and structure analyses. This database is publicly available at https://github.com/liskinsk/Aldolase-variant-and-sequence-database.

Pharmacokinetics and apparent Michaelis constant for metabolite conversion of sorafenib in healthy and hepatocellular carcinoma-bearing rats.

Aim: Investigation of the pharmacokinetics of sorafenib (SRF) in rats with hepatocellular carcinoma (HCC). Methods: A reproducible ultra-HPLC-MS method for simultaneous determination of serum SRF, N-hydroxymethyl sorafenib and N-demethylation sorafenib. Results: Both the maximum serum concentrations (2.5-times) and the area under the serum concentration-time curve from 0 h to infinity (4.5-times) of SRF were observed to be significantly higher, with a greater than 3.0-fold decrease in the clearance rate in the HCC-bearing rats compared with these values in healthy animals. Further study revealed approximately 3.8- and 3.2-times increases in the apparent Michaelis constant for N-hydroxymethyl sorafenib and N-demethylation sorafenib conversions in the HCC-bearing rats. Conclusion: The low efficiency for the SRF conversions was a key contributor to the increased serum concentrations of SRF.

Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells

Detection of superoxide anion (O2·-) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2·-. The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10−9 mol cm−2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+) catalyzes the dismutation of O2·- to O2 and H2O2, and H2O2 is then electrochemically oxidized to generate amperometric signals for determination of O2·-. The sensor demonstrates outstanding detection performance for O2·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM–1.34 μM and 1.34 μM–42.97 μM) with corresponding sensitivity of 0.479 and 0.052 μA μM−1cm−2, respectively. Additionally, the calculated apparent Michaelis constant (Kmapp) of 1.804 μM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.

Abstract 17158: Effects of Human Monoclonal Antibodies on ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Introduction: Immune thrombotic thrombocytopenic purpura (iTTP) is caused by antibody-mediated deficiency of the metalloprotease ADAMTS13, which cleaves VWF. Myocardial infarction and ischemic strokes are common and often fatal sequelae of iTTP. Inhibitory anti-ADAMTS13 antibodies appear to affect catalytic turnover more than apparent substrate binding affinity (PMID 37023370). Exploring how antibodies affect VWF cleavage by ADAMTS13 in physiologic conditions is needed to better understand the pathophysiology of iTTP. Hypothesis: Anti-ADAMTS13 antibodies may influence ADAMTS13 function by affecting catalytic turnover and/or apparent substrate binding affinity differently in physiologic conditions than in standard assay conditions. Aims: Characterize how single chain fragments of the variable loop (scFv’s) previously identified from a phage display library derived from several iTTP patients’ antibodies affect ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and see if/how they differ from scFv-mediated effects in standard conditions. Methods: ADAMTS13 in normal human plasma was titrated with increasing concentrations of the substrate FRETS-VWF73 to derive maximum velocity ( V max ) and apparent Michaelis constant ( K M,app ) in standard assay conditions (pH 6.0) and conditions closer to physiologic (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv’s. Results: Compared to VWF cleavage by ADAMTS13 in the absence of scFv, the inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0; K M,app decreased at pH 6.0 and increased at pH 7.0. The stimulatory scFv3-3 and scFv4-41 increased V max at each pH, while K M,app in the presence of each scFv decreased only at pH 7.0. Conclusions: Anti-ADAMTS13 antibody-mediated effects on VWF cleavage differ when comparing standard and more physiologic pH conditions, guiding future investigative directions into iTTP pathophysiology.

Thermodynamic Effects of Antibodies on VWF73 Cleavage By ADAMTS13

Background and objective: Immune thrombotic thrombocytopenic purpura (iTTP) is an antibody-mediated life-threatening disease, caused by severe deficiency a plasma metalloprotease ADAMTS13 that impairs the cleavage of ultra-large von Willebrand factor (VWF). This leads to catastrophic microvascular thrombosis and angiopathic hemolysis. Exploring the mechanism of antibodies that inhibit ADAMTS13 under physiologic conditions and standard assay conditions for diagnosis of iTTP (pH 6.0) may help better understand the pathophysiology of iTTP. Our previous study suggests that an inhibitory anti-ADAMTS13 antibody may affect catalytic turnover of the enzyme more than its apparent substrate binding affinity. Here, we describe a rigorous method to quantitatively determine thermodynamic linkage between antibody binding and substrate binding affinity under various conditions. We used well-characterized single chain fragments of the variable loop (scFv's) identified from a phage display library derived from several iTTP patients, and compare the apparent Michaelis constant ( K M,app,) and the maximal enzyme velocity ( V max) of ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and in standard conditions. We also describe the stimulatory effect of a newly characterized scFv that targets the C-terminus of ADAMTS13. Methods: Single chain fragments of the variable region (scFv4-20, scFv3-3 and scFv4-41), isolated by phage display from a human anti-ADAMTS13 antibody library derived from iTTP patients, were expressed and purified. ADAMTS13 in normal human plasma was titrated with increasing concentrations of the fluorescently labeled surrogate substrate FRETS-VWF73 to derive V maxand K M,appin the standard assay conditions (pH 6.0) and the conditions closer to physiologic pH (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv's. Reaction quotient (Q), which defines thermodynamic linkage between an allosteric effector and substrate, is derived from the ratio of K M,app when saturating scFv is present and K M,app without antibodies. Results: The V max of VWF73 cleavage by ADAMTS13 was lower at pH 7.0 than at pH 6.0 (5.23 ± 0.15 ΔFU/sec vs 7.48 ± 0.42 ΔFU/sec, respectively), and K M,app at pH 6.0 (2.54 ± 0.26 µM) was higher than at pH 7.0 (1.48 ± 0.11 µM) in the absence of scFv. Saturating inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0 (4.45 ± 0.16 ΔFU/sec and 1.00 ± 0.14 ΔFU/sec, respectively); K M,app was relatively unaffected at pH 6.0 (2.70 ± 0.07 µM, Q 1.06) and decreased at pH 7.0 (1.08 ± 0.27 µM, Q 0.73). Saturating scFv3-3 and scFv4-41 increased V max at both pH 6.0 (9.86 ± 0.46 ΔFU/sec and 10.05 ± 0.67 ΔFU/sec, respectively) and 7.0 (9.09 ± 0.25 ΔFU/sec and 10.26 ± 0.34 ΔFU/sec, respectively); while in the presence of scFv3-3 and scFv4-41, K M,app decreased slightly at pH 6.0 (2.20 ± 0.08 µM, Q 0.87; and 2.36 ± 0.26 µM, Q 0.93, respectively) and more dramatically at pH 7.0 (1.08 ± 0.05 µM, Q 0.73; and 1.16 ± 0.07 µM, Q 0.78, respectively). (Figure 1). Conclusions: These results indicate that anti-ADAMTS13 IgG-mediated effects on VWF cleavage may differ when comparing the standard and more physiologic pH conditions. At pH 7.0, both inhibitory and stimulatory scFvs exert a stronger effect on V max of VWF73 cleavage by ADAMTS13 than at pH 6.0. The scFv4-41, which was not previously characterized, had a stimulatory effect on ADAMTS13 very similar to the previously characterized scFv3-3. The K M,app decreases with the increasing of pH from 6.0 to 7.0, while the presence of saturated scFvs investigated in the study appears to increase apparent substrate binding affinity in conditions close to physiologic. The change of enzyme kinetics of VWF73 cleavage by ADAMTS13 in presence of saturated antibodies adds the understanding of iTTP pathophysiology and guide future investigative directions. Key words: Immune thrombotic thrombocytopenic purpura; ADAMTS13, inhibitory and stimulatory antibody

Enzymatic properties and clinical associations of serum alpha-galactosidase A in Parkinson's disease.

Recent studies have revealed an association between Parkinson's disease (PD) and Fabry disease, a lysosomal storage disorder; however, the underlying mechanisms remain to be elucidated. This study aimed to investigate the enzymatic properties of serum alpha-galactosidase A (GLA) and compared them with the clinical parameters of PD. The study participants consisted of 66 sporadic PD patients and 52 controls. We measured serum GLA activity and calculated the apparent Michaelis constant (Km ) and maximal velocity (Vmax ) by Lineweaver-Burk plot analysis. Serum GLA protein concentration was measured by enzyme-linked immunosorbent assay. We examined the potential correlations between serum GLA activity and GLA protein concentration and clinical features and the plasma neurofilament light chain (NfL) level. Compared to controls, PD patients showed significantly lower serum GLA activity (P < 0.0001) and apparent Vmax (P = 0.0131), but no change in the apparent Km value. Serum GLA protein concentration was lower in the PD group (P = 0.0168) and was positively associated with GLA activity. Serum GLA activity and GLA protein concentration in the PD group showed a negative correlation with age. Additionally, serum GLA activity was negatively correlated with the motor severity score and the level of plasma NfL, and was positively correlated with the score of frontal assessment battery. This study highlights that the lower serum GLA activity in PD is the result of a quantitative decrement of GLA protein in the serum and that it may serve as a biomarker of disease severity.

Electrochemical and biosensing properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens

We investigated the bioelectrochemical properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens (TvGDH) and its electrochemical behaviour when immobilized on a graphite electrode. TvGDH was recently shown to have an unusual substrate spectrum and to prefer maltose over glucose as substrate, and hence could be of interest as recognition element in a maltose sensor. In this study, we determined the redox potential of TvGDH, which is –0.268 ± 0.007 V vs. SHE, and advantageously low to be used with many redox mediators or redox polymers. The enzyme was entrapped in, and wired by an osmium redox polymer (poly(1-vinylimidazole-co-allylamine)-{[Os(2,2′-bipyridine)2Cl]Cl}) with formal redox potential of +0.275 V vs. Ag|AgCl via poly(ethylene glycol) diglycidyl ether crosslinking onto a graphite electrode. When the TvGDH-based biosensor was tested with maltose it showed a sensitivity of 1.7 μA mM−1cm−2, a linear range of 0.5–15 mM, and a detection limit of 0.45 mM. Furthermore, it gave the lowest apparent Michaelis-Menten constant (KM app) of 19.2 ± 1.5 mM towards maltose when compared to other sugars. The biosensor is also able to detect other saccharides including glucose, maltotriose and galactose, these however also interfere with maltose sensing.

Direct Electrochemistry of Glucose Dehydrogenase-Functionalized Polymers on a Modified Glassy Carbon Electrode and Its Molecular Recognition of Glucose.

A glucose biosensor was layer-by-layer assembled on a modified glassy carbon electrode (GCE) from a nanocomposite of NAD(P)+-dependent glucose dehydrogenase, aminated polyethylene glycol (mPEG), carboxylic acid-functionalized multi-wall carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymers. The electrochemical electrode was denoted as NF/IL/GDH/mPEG-fMWCNTs/GCE. The composite polymer membranes were characterized by cyclic voltammetry, ultraviolet-visible spectrophotometry, electrochemical impedance spectroscopy, scanning electron microscopy, and transmission electron microscopy. The cyclic voltammogram of the modified electrode had a pair of well-defined quasi-reversible redox peaks with a formal potential of -61 mV (vs. Ag/AgCl) at a scan rate of 0.05 V s-1. The heterogeneous electron transfer constant (ks) of GDH on the composite functional polymer-modified GCE was 6.5 s-1. The biosensor could sensitively recognize and detect glucose linearly from 0.8 to 100 µM with a detection limit down to 0.46 μM (S/N = 3) and a sensitivity of 29.1 nA μM-1. The apparent Michaelis-Menten constant (Kmapp) of the modified electrode was 0.21 mM. The constructed electrochemical sensor was compared with the high-performance liquid chromatography method for the determination of glucose in commercially available glucose injections. The results demonstrated that the sensor was highly accurate and could be used for the rapid and quantitative determination of glucose concentration.

Fabrication of glucose bioelectrochemical sensor based on Au@Pd core-shell supported by carboxylated graphene oxide

The study presents a novel electrochemical glucose biosensor based on glucose oxidase (GOx) immobilized on Au@Pd core-shell nanoparticles supported on carboxylated graphene oxide (cGO). The immobilization of GOx was achieved by cross-linking the chitosan biopolymer (CS) including Au@Pd/cGO and glutaraldehyde (GA) on a glassy carbon electrode. The analytical performance of GCE/Au@Pd/cGO-CS/GA/GOx was investigated using amperometry. The biosensor had fast response time (5.2 ± 0.9 s), a satisfactory linear determination range between 2.0 × 10−5 and 4.2 × 10−3 M, and limit of detection of 10.4 μM. The apparent Michaelis-Menten constant (Kapp) was calculated as 3.04 mM. The fabricated biosensor also exhibited good repeatability, reproducibility, and storage stability. No interfering signals from dopamine, uric acid, ascorbic acid, paracetamol, folic acid, mannose, sucrose, and fructose were observed. The large electroactive surface area of carboxylated graphene oxide is a promising candidate for sensor preparation.

A highly sensitive MXene/AuPt/AChE-based electrochemical platform for the detection of chlorpyrifos

The MXene/AuPt nanocomposite was successfully synthesized through the reduction process, which possessed good biocompatibility and electron transfer capability. On the basis of the excellent performance of MXene/AuPt and the enzyme inhibition, an ultrasensitive and high-performance acetylcholinesterase (AChE)-based electrochemical biosensor was prepared for the detection of chlorpyrifos. The prepared biosensor combined the unique electrocatalytic property and synergistic effect of MXene nanosheets and AuPt bimetallic nanoparticles, which not only promoted the electron transfer capability, but also increased the electroactive surface area for the immobilization of AChE. The electrochemical performance was investigated by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. This biosensor exhibited good affinity towards AChE with a corresponding apparent Michaelis constant (Km) of 1.27 mM. Under optimal conditions, the electrochemical biosensing platform presented a good linear relationship in the range of 10−8−10−3 mg mL−1 and a low limit of detection of 1.55 pg mL−1. Furthermore, the as-prepared electrochemical biosensor exhibited high sensitivity, acceptable repeatability, and excellent stability, which implied the great potential for the detection of organophosphorus pesticides.

Intestinal in vitro transport assay combined with physiologically based kinetic modeling as a tool to predict bile acid levels in vivo.

Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. We used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis-Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app), and ODE’s inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated into a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. ODE inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels.

Evaluation of Cotton Seeds as Environmentally Liable Source for Neutral Protease

Proteases are considered one of the most imperative groups of enzymes and are used in bioremediation processes, leather, detergents, pharmaceutical and the food industry. The foremost aim of this study was to extract, purify, and characterize the protease enzyme from cotton seeds. Purification of the neutral protease was achieved with ammonium sulphate, which gave the best results at 60% concentration with a specific activity of 51 units/mg protein and 1.52 purification fold with a percentage yield of 10.5%. The protease was active and stable at a wide range of pH from 4.0–10.0 with an optimum pH of 7.0. The highest activity of the purified enzyme was found at 20 °C. The enzyme was thermally stable and retained 25% of its activity at 50o C. The activity of cottonseeds protease Fraction-IV was enhanced by 20% with ZnCl2 and 15% with CoCl2 as enzyme samples were heated for 10 minutes. The Casein and peptone assays were also performed to check its catalytic activity. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 19 μmol/min and 0.08 mol/L, respectively, while activation energy was found to be 12.47 KJ/mol.

Çok Duvarlı Karbon Nanotüpler/Politiyofen Kompozit Kullanılarak Hazırlanan Amperometrik Glikoz Biyosensörü

In this study, multi-walled carbon nanotubes/polythiophene composite (MWCNTs/PTh) modified glassy carbon electrode was used for the amperometric detection of glucose. Glucose oxidase (GOx) was entrapped by a crosslinking agent on the MWCNTs/PTh composite film synthesized by electrochemical polymerization of thiophene onto MWCNTs. Characterization of composite film was achieved by cyclic voltammetry (CV), fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) techniques. The amperometric measurements of electrode was performed at +0.70V vs. SCE, which was the electrooxidation potential of enzymatically produced H2O2. The effects of thiophene amount in the composite, pH, temperature and substrate concentration were investigated on the response of enzyme electrode. Optimum pH was 7.0 at room temperature and the response time of enzyme electrode was 25 s. The upper limit of the linear working range was 4.85 mM glucose concentration. The limit of detection of sensor was calculated as 148 µM. The sensitivity of glucose biosensor was determined as 4.39 µA mM-1 cm-2. The value of apparent Michaelis-Menten constant (KMapp) was 1.68 mM according to the Lineweaver-Burk equation. The activation energy of this immobilized enzyme system was 88.92 kJ mol-1.

Immobilization of Glucose Oxidase on Glutathione Capped CdTe Quantum Dots for Bioenergy Generation

An efficient immobilization of Glucose oxidase (GOx) on an appropriate substrate is one of the main challenges of developing fuel cells that allow energy to be obtained from renewable substrates such as carbohydrates in physiological environments. The research importance of biofuel cells relies on their experimental robustness and high compatibility with biological organisms such as tissues or the bloodstream with the aim of obtaining electrical energy even from living systems. In this work, we report the use of 5,10,15,20 tetrakis (1-methyl-4-pyridinium) porphyrin and glutathione capped CdTe Quantum dots (GSH-CdTeQD) as a support matrix for the immobilization of GOx on carbon surfaces. Fluorescent GSH-CdTeQD particles were synthesized and their characterization by UV-Vis spectrophotometry showed a particle size between 5–7 nm, which was confirmed by DLS and TEM measurements. Graphite and Toray paper electrodes were modified by a drop coating of porphyrin, GSH-CdTeQD and GOx, and their electrochemical activity toward glucose oxidation was evaluated by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Additionally, GOx modified electrode activity was explored by scanning electrochemical microscopy, finding that near to 70% of the surface was covered with active enzyme. The modified electrodes showed a glucose sensitivity of 0.58 ± 0.01 μA/mM and an apparent Michaelis constant of 7.8 mM. The addition of BSA blocking protein maintained the current response of common interferent molecules such as ascorbic acid (AA) with less than a 5% of interference percentage. Finally, the complex electrodes were employed as anodes in a microfluidic biofuel cell (μBFC) in order to evaluate the performance in energy production. The enzymatic anodes used in the μBFC allowed us to obtain a current density of 7.53 mAcm−2 at the maximum power density of 2.30 mWcm−2; an open circuit potential of 0.57 V was observed in the biofuel cell. The results obtained suggest that the support matrix porphyrin and GSH-CdTeQD is appropriate to immobilize GOx while preserving the enzyme’s catalytic activity. The reported electrode arrangement is a viable option for bioenergy production and/or glucose quantification.

Enzymes Encapsulated within Alginate Hydrogels: Bioelectrocatalysis and Electrochemiluminescence Applications.

A simple procedure to incorporate enzymes (horseradish peroxidase, HRP, and lactate oxidase, LOx) within alginate hydrogels is reported with electrochemiluminescence (ECL) used to detect the enzymatic reactions with the corresponding substrates. First, HRP and LOx were successfully immobilized into CaCO3 microspheres, followed by the electrostatic layer-by-layer deposition of a nanoshell onto the microspheres, and finally by their dispersion into alginate solution. The as-prepared dispersion was drop cast onto the glassy carbon electrodes and cross-linked by the external and internal gelation methods using Ca2+ cations. The enzymes encapsulated within the alginate hydrogels were characterized using cyclic voltammetry and kinetic studies performed using ECL. The results showed that the enzymatic activity was significantly maintained as a result of the immobilization, with values of the apparent Michaelis-Menten constants estimated as 7.71 ± 0.62 and 8.41 ± 0.43 μM, for HRP and LOx, respectively. The proposed biosensors showed good stability and repeatability with an estimated limit of detection of 5.38 ± 0.05 and 0.50 ± 0.03 μM for hydrogen peroxide and lactic acid, respectively. The as-prepared enzymes encapsulated within the alginate hydrogels showed good stability up to 28 days from their preparation. The sensitivity and selectivity of the enzymes encapsulated within the alginate hydrogels were tested in real matrices (HRP, hydrogen peroxide, in contact lens solution; LOx, lactic acid in artificial sweat) showing the sensitivity of the ECL detection methods for the detection of hydrogen peroxide and lactic acid in real samples.

The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase.

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Isolation and Purification of Bacillus amyloliquefaciens D1 Protease and Its Application in the Fermentation of Soybean Milk to Produce Large Amounts of Free Amino Acids.

In this study, a strain of Bacillus amyloliquefaciens D1, with a notably high production of neutral protease, was isolated from Morchella crassipes. The protease was purified to 10.4-fold with a specific activity of 4542.9 U/mg and 2.7% recovery. The enzyme was purified by 70% (NH4)2SO4 and DEAE-Cellulose-52 column. The estimated molecular mass of the purified protease obtained by SDS-PAGE was approximately 40kDa. The enzyme was optimally active at pH 6.0 and 50°C. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) values of the purified protease were 8.2mg/mL and 65.7µg/(min mL). The enzymatic properties and rapid and efficient purification of Bacillus amyloliquefaciens D1 provide the basis for its potential commercialization and industrial development. Moreover, more essential amino acids, such as isoleucine, leucine, and phenylalanine, would be released when the strain fermented soybean milk, and then a better amino acid profile would be formed in soybean milk. Results suggest that this strain exhibits great potential in fermented soybean milk, and the enzyme could lay a foundation for its industrial application and further research.

Enzyme Electrode Biosensors for N-Hydroxylated Prodrugs Incorporating the Mitochondrial Amidoxime Reducing Component.

Human mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) were immobilised on glassy carbon electrodes using the crosslinker glutaraldehyde. Voltammetry was performed in the presence of the artificial electron transfer mediator methyl viologen, whose redox potential lies negative of the enzymes' MoVI/V and MoV/IV redox potentials which were determined from optical spectroelectrochemical and EPR measurements. Apparent Michaelis constants obtained from catalytic limiting currents at various substrate concentrations were comparable to those previously reported in the literature from enzymatic assays. Kinetic parameters for benzamidoxime reduction were determined from cyclic voltammograms simulated using Digisim. pH dependence and stability of the enzyme electrode with time were also determined from limiting catalytic currents in saturating concentrations of benzamidoxime. The same electrode remained active after at least 9 days. Fabrication of this versatile and cost-effective biosensor is effective in screening new pharmaceutically important substrates and mARC inhibitors.

Mutational analysis of compound heterozygous mutation p.Q6X/p.H232R in SRD5A2 causing 46,XY disorder of sex development

BackgroundOver 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration.MethodsThe two 46,XY DSD cases were identified and sequenced. In order to identify the source of the compound heterozygous p.Q6X/p.H232R mutation, the parents, maternal grandparents, and maternal uncle were sequenced. Since p.Q6X mutation is a nonsense mutation, p.H232R mutation was transfected into HEK293 cells and dihydrotestosterone (DHT) production were analyzed by liquid chromatography–mass spectrometry (LC–MS) for 5α-reductase 2 enzyme activities test. Apparent michaelis constant (Km) were measured of p.H232R mutation to analyze the binding ability change of 5α-reductase 2 enzyme with testosterone (T) or NADPH.ResultsThe sequence results showed that the two 46,XY DSD cases were the compound heterozygous p.Q6X/p.H232R mutation, of which the heterozygous p.Q6X mutation originating from maternal family and heterozygous p.H232R mutation originating from the paternal family. The function analysis confirmed that p.H232R variant decreased the DHT production by LC–MS test. The Km analysis demonstrated that p.H232R mutation affected the binding of SRD5A2 with T or NADPH.ConclusionsOur findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

A sensitive and stable acetylcholinesterase biosensor with TiO2 nanoparticles anchored on graphitic carbon nanofibers for determination of organophosphate pesticides.

Electrode materials play a central role in assembling biosensors. In this work, a titanium dioxide nanoparticle loaded graphitized carbon nanofiber (TiO2/GNF) composite is prepared for the sensitive detection of organophosphorus pesticide residues (OPs). The TiO2/GNF composite with superior conductivity, catalytic activity and biocompatibility offers an extremely hydrophilic surface for the effective immobilization of acetylcholinesterase (AChE). Furthermore, the Ti atoms of TiO2/GNFs could coordinate with AChE to improve its stability, and TiO2 has a strong adsorption on OPs. The developed AChE/TiO2/GNFs/GCE biosensor showed a high affinity to acetylthiocholine chloride (ATCh) and could catalyze the hydrolysis of ATCh with an apparent Michaelis-Menten constant (Km) of 50 μM. The constructed AChE/TiO2/GNFs/GCE biosensor exhibits a wide detection linear range (1.0 × 10-13 M to 1.0 × 10-8 M) with a low detection limit (3.3 fM) for paraoxon determination (a model of OPs). In addition, the developed biosensor possesses remarkable anti-interference, acceptable reproducibility and good long-term stability, and is successfully used for the determination of OPs in lake water, providing a new strategy for the analysis of OPs in ecological environments.