Inactivation Of Urease Research Articles

Biochemical and thermodynamic properties of de novo synthesized urease from Vicia sativa seeds with enhanced industrial applications

Urease is one of the most significant enzymes in the industry. The objective of this research was to isolate and partially purify urease from Vicia sativa seeds with urease characterization. With a 6.4 % yield, the purification fold was 9.0. By using chromatography, it was determined that the isolated urease had a molecular weight of 55 kDa. The maximum urease activity was found following a 60-s incubation period at 40 °C and pH 8. The activity of urease was significantly boosted by a mean of calcium, barium, DL-dithiothreitol, Na2EDTA, and citrate (16.9, 26.6, 18.6, 13.6, and 31 %), respectively. But nickel and mercury caused inhibitory effects and completely inhibited urease activity, indicating the presence of a thiol (-SH) group in the enzyme active site. The Arrhenius plot was used to analyze the thermodynamic constants of activation, Ea, ΔH*, ΔG*, and ΔS*. The results showed that the values were 30 kJ/mol, 93.14 kJ/mol, 107.17 kJ/mol/K, and −40.80 J/mol/K, respectively. The significance of urease extraction from various sources may contribute to our understanding of the metabolism of urea in plants. The current report has novelty as it explained for the first time the kinetics and thermodynamics of hydrolysis of urea and inactivation of urease from V. sativa seeds.

Enzymatic Nanomotors Surviving Harsh Conditions Enabled by Metal Organic Frameworks Encapsulation.

Enzyme-driven micro/nanomotors (MNMs) have demonstrated potentials in the biomedical field because of their excellent biocompatibility, versatility, and fuel bioavailability. However, the fragility of enzymes limits their practical application, because of their susceptibility to denaturation and degradation in realistic scenarios. Herein, a simple yet versatile and effective approach is reported to preserve the enzymatic activity and propulsion capability of enzymatic MNMs under various harsh conditions using metal organic frameworks (MOFs) as a protective shell. Urease can be encapsulated within the exoskeleton of zeolitic imidazolate framework-8 (ZIF-8) via biomimetic mineralization to form ZIF-8@urease (ZU-I) nanomotors that exhibit self-propulsion in the presence of urea. When exposed to harsh conditions, including high temperature, presence of proteases, and organic solvents, the ZU-I nanomotors still maintained their activity and mobility, whereas ZIF-8 with externally modified urease (ZU-O) nanomotors with externally modified urease as a control rapidly lost their motion capabilities owing to the inactivation of urease. Furthermore, ZU-I nanomotors exhibit effectively enhanced diffusion within the small intestine fluid, achieving a fourfold higher mucus penetration than the ZU-O nanomotors. The results highlight the effectiveness of using MOFs as protective shells for enzyme nano-engines, which can greatly advance the practical applications of enzymatic MNMs under realistic conditions, especially for biomedical purpose.

Kinetic and structural details of urease inactivation by thiuram disulphides

This paper reports on the molecular details of the reactivity of urease, a nickel-dependent enzyme that catalyses the last step of organic nitrogen mineralization, with thiuram disulphides, a class of molecules known to inactivate the enzyme with high efficacy but for which the mechanism of action had not been yet established. IC50 values of tetramethylthiuram disulphide (TMTD or Thiram) and tetraethylthiuram disulphide (TETD or Disulfiram) in the low micromolar range were determined for plant and bacterial ureases. The X-ray crystal structure of Sporosarcina pasteurii urease inactivated by Thiram, determined at 1.68 Å resolution, revealed the presence of a covalent modification of the catalytically essential cysteine residue. This is located on the flexible flap that modulates the size of the active site channel and cavity. Formation of a Cys-S-S-C(S)-N(CH3)2 functionality responsible for enzyme inactivation was observed. Quantum-mechanical calculations carried out to rationalise the large reactivity of the active site cysteine support the view that a conserved histidine residue, adjacent to the cysteine in the active site flap, modulates the charge and electron density along the thiol SH bond by shifting electrons towards the sulphur atom and rendering the thiol proton more reactive. We speculate that this proton could be transferred to the nickel-coordinated urea amide group to yield a molecule of ammonia from the generated Curea-NH3+ functionality during catalysis.

Gemifloxacin-transition metal complexes as therapeutic candidates: antimicrobial, antifungal, anti-enzymatic, and docking studies of newly synthesized complexes

In the era of acquired microbial resistance (AMR), resulting in the ineffectiveness of antibiotics is of keen interest for researchers in current scenarios. Ten novel metal complexes of gemifloxacin have been synthesized by reacting it with essential and trace elements in a 2:1 ratio predetermined conducto-metrically. As these metals are either present in the body or co-administered as metallic supplements can alter the level of antibiotics. Therefore, Metal complexes of Gemifloxacin, an important member of the fluoroquinolone family, were synthesized. The possible coordination of gemifloxacin with these metals has been proposed by the electronic and elemental data obtained through molar conductance, elemental analysis, and spectroscopic techniques like ultraviolet-visible (UV-Vis), infrared (IR), and proton-nuclear magnetic resonance (1H NMR) studies.In the light of these studies, the monoanionic bidentate ligand behavior of gemifloxacin in complexation with metals has been revealed. For in-vitro microbial studies, these newly synthesized complexes were tested against eleven different bacteria including Gram + ve and Gram -ve organisms, and one fungal strain. The results were compared with the parent drug by applying ANOVA through SPSS software version 22. Therefore, it has been found that among all synthesized metal complexes, the G-M01 complex exhibits increased activity against B. subtilis, P. mirabilis, E. coli, K. pneumonia, and C. freundii. Complex G-M02, G-M03, G-M04, and G-M10 show more pronounced activity than Gemifloxacin against S. aureus and M. luteus. Moreover, the binding orientations of the synthesized metal complexes into the binding site of the urease enzyme revealed that all the docked metal complexes oriented away from the Ni bi-center, and the inactivation of urease is due to their interaction with entrance flap residues.

Inactivation of Jack Bean Urease by Nitidine Chloride from Zanthoxylum nitidum: Elucidation of Inhibitory Efficacy, Kinetics and Mechanism.

Urease is a metalloenzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide, which has a negative impact on human health and agriculture. In this study, the inactivation of jack bean urease by nitidine chloride (NC) was investigated to elucidate the inhibitory effect, kinetics, and underlying mechanism of action. The results showed that NC acted as a concentration- and time-dependent inhibitor with an IC50 value of 33.2 ± 4.8 μM and exhibited a similar inhibitory effect to acetohydroxamic acid (IC50 = 31.7 ± 5.8 μM). Further kinetic analysis demonstrated that NC was a slow-binding and non-competitive inhibitor for urease. Thiol-blocking reagents (dithiothreitol, glutathione, and l-cysteine) significantly retarded urease inactivation, while Ni2+ competitive inhibitors (boric acid and sodium fluoride) synergetically suppressed urease with NC, suggesting that the active site sulfhydryl groups were possibly obligatory for NC blocking urease. Molecular docking simulation further argued its inhibition mechanism. Additionally, NC-induced deactivation of urease was verified to be reversible since the inactivated enzyme could be reactivated by glutathione. Taking together, NC was a non-competitive inhibitor targeting the thiol group at the active site of urease with characteristics of concentration dependence, reversibility, and slow binding, serving as a promising novel urease suppressant.

Biological soil activity when applying liquid manure on rice fields in Kuban

The research was aimed at studying the effect of liquid manure application on biological activity of meadow-chernozem soil (GleyicChernozem) when growing rice on the Maryano-Cheburgol irrigation massif of the rice irrigation system of the Krasnoarmeisky district of the Krasnodar Territory. The experimental scheme included the following options: without fertilizers (control); rice cultivation technology on a farm; autumn manure pumpapplication (30 t/ha); autumn manure pump application (30 t/ha) + Entec FL DMPP nitrification inhibitor; spring manure pumpapplication (30 t/ha); spring manure pumpapplication (30 t/ha) + Entec FL DMPP nitrification inhibitor. It was found that the autumn application of liquid manure into the soil combined with the Entec nitrification inhibitor contributed to an increase in the amount of CO2 emitted from the soil by 0.031–0.038 mg CO2 kg−1 day−1; inactivation of urease in the germination phase and its activation during rice tillering, which ensured a longer preservation of nitrogen in the soil; an increase in catalase activity during the period of maximum peroxide formation (in the rice flowering phase) by 11–29% and invertase activity by 2.3–7.5% compared with other options fertilized with manure.

Inhibition of Urease, a Ni‐Enzyme: The Reactivity of a Key Thiol With Mono‐ and Di‐Substituted Catechols Elucidated by Kinetic, Structural, and Theoretical Studies

AbstractThe inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by a class of six aromatic poly‐hydroxylated molecules, namely mono‐ and dimethyl‐substituted catechols, was investigated on the basis of the inhibitory efficiency of the catechol scaffold. The aim was to probe the key step of a mechanism proposed for the inhibition of SPU by catechol, namely the sulfanyl radical attack on the aromatic ring, as well as to obtain critical information on the effect of substituents of the catechol aromatic ring on the inhibition efficacy of its derivatives. The crystal structures of all six SPU‐inhibitors complexes, determined at high resolution, as well as kinetic data obtained on JBU and theoretical studies of the reaction mechanism using quantum mechanical calculations, revealed the occurrence of an irreversible inactivation of urease by means of a radical‐based autocatalytic multistep mechanism, and indicate that, among all tested catechols, the mono‐substituted 3‐methyl‐catechol is the most efficient inhibitor for urease.

Inhibition of Urease, a Ni-Enzyme: The Reactivity of a Key Thiol With Mono- and Di-Substituted Catechols Elucidated by Kinetic, Structural, and Theoretical Studies.

The inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by a class of six aromatic poly-hydroxylated molecules, namely mono- and dimethyl-substituted catechols, was investigated on the basis of the inhibitory efficiency of the catechol scaffold. The aim was to probe the key step of a mechanism proposed for the inhibition of SPU by catechol, namely the sulfanyl radical attack on the aromatic ring, as well as to obtain critical information on the effect of substituents of the catechol aromatic ring on the inhibition efficacy of its derivatives. The crystal structures of all six SPU-inhibitors complexes, determined at high resolution, as well as kinetic data obtained on JBU and theoretical studies of the reaction mechanism using quantum mechanical calculations, revealed the occurrence of an irreversible inactivation of urease by means of a radical-based autocatalytic multistep mechanism, and indicate that, among all tested catechols, the mono-substituted 3-methyl-catechol is the most efficient inhibitor for urease.

Kinetic and structural analysis of the inactivation of urease by mixed-ligand phosphine halide Ag(I) complexes

Soft metal ions can inactivate urease, a Ni(II)-dependent enzyme whose hydrolytic activity has significant implications in agro-environmental science and human health. Kinetic and structural studies of the reaction of Canavalia ensiformis urease (JBU) and Sporosarcina pasteurii urease (SPU) with Ag(I) compounds of general formula [Ag(PEt3)X]4 (X = Cl, Br, I), and with the ionic species [Ag(PEt3)2]NO3, revealed the role of the Ag(I) ion and its ligands in modulating the metal-enzyme interaction. The activity of JBU is obliterated by the [Ag(PEt3)X]4 complexes, with IC50 values in the nanomolar range; the efficiency of the inhibition increases in the Cl− < Br− < I− order. The activity of JBU upon [Ag(PEt3)2]NO3 addition decreases to a plateau corresponding to ca. 60% of the original activity and decreases with time at a reduced rate. Synchrotron X-ray crystallography on single crystals obtained after the incubation of SPU with the Ag(I) complexes yielded high-resolution (1.63–1.97 Å) structures. The metal-protein adducts entail a dinuclear Ag(I) cluster bound to the conserved residues αCys322, αHis323, and αMet367, with a bridging cysteine thiolate atom, a weak Ag…Ag bond, and a quasi-linear Ag(I) coordination geometry. These observations suggest a mechanism that involves the initial substitution of the phosphine ligand, followed by a structural rearrangement to yield the dinuclear Ag(I) cluster. These findings indicate that urease, in addition to the active site dinuclear Ni(II) cluster, possesses a secondary metal binding site, located on the mobile flap domain, capable of recognizing pairs of soft metal ions and controlling catalysis.

Temperature‐dependent inactivation and catalysis rates of plant‐based ureases for engineered biomineralization

AbstractEngineered (bio)mineralization uses the enzyme urease to catalyze the hydrolysis of urea to promote carbonate mineral precipitation. The current study investigates the influence of temperature on ureolysis rate and degree of inactivation of plant‐sourced ureases over a range of environmentally relevant temperatures. Batch experiments at 30°C demonstrated that jack bean meal (JBM) has a 1.7 to 56 times higher activity (844 μmol urea hydrolyzed g−1 JBM min−1) than the other tested plant‐sourced ureases (soybean, pigeon pea and cottonseed). Hence, ureolysis and enzyme inactivation rates were evaluated for JBM at temperatures between 20°C and 80°C. A combined first‐order urea hydrolysis and first‐order enzyme inactivation model described the inactivation of urease over the investigated range of temperatures. The temperature‐dependent rate coefficients (kurea) increased with temperature and ranged from 0.0018 at 20°C to 0.0249 L g−1 JBM min−1 at 80°C; JBM urease became ≥50% inactivated in as little as 5.2 minutes at 80°C and in as long as 2238 minutes at 50°C. The combined urea hydrolysis kinetics and enzyme inactivation model provides a mathematical relationship useful for the design of biomineralization technologies and can be incorporated into reactive transport models.

Modeling of urease thermal inactivation processes in soybean at high-temperature micronization

The use of soybean, in particular in forage production without preliminary heat treatment is not appropriate, and sometimes dangerous, because of the presence of antinutrients. As a marker in assessing safety of cakes and meals, there is often used urease in forage production. This paper describes the results of thermal inactivation of urease in soybean during the process of high-temperature micronization (heating of grain in the flux of infrared radiation). There have been obtained the empirical dependencies of the degree of its inactivation on time of heat treatment and energy exposure (the product of irradiation by the time of treatment). The similar dependences of urease activity on grain temperature are invariant to infrared heating (irradiation and time) regimes, but their nature is affected by the initial moisture content. The paper proposes the models of inactivation of antinutrients based on of the first-order equations of chemical kinetics with the reaction rate constant in various forms (Arrhenius and Hinshelwood, the transition state theory). The models have been tested on literature data on the inactivation of a trypsin inhibitor at a constant temperature. The models are further refined taking into account the variable (increasing) temperature and are reduced to the simplest form: Y= k [Exp (-εR/T) - T0 еxp (-εR/T0)], where T, T0- are the current and initial temperatures of grain, k, εR - theempirical coefficients. The identification of the model coefficients was carried out based on the results of inactivation of urease during heating in the flux of infrared radiation. It has been established that the results of thermal inactivation of soybean do not depend on the IR processing regimes and are determined only by the initial moisture content of grain, and by the end heating temperature. The efficiency of inactivation is higher the higher is the used irradiation. There is a compensating effect - with the growth in one coefficient, another is also increased. The considered models can be used for the thermal degradation processes and other thermolabile substances.

An Evaluation of Maleic‐Itaconic Copolymers as Urease Inhibitors

Core Ideas Maleic‐Itaconic Polymers (MIPs) strongly and rapidly inactivate urease at pH 5.0. MIPs favour the release of the essential Ni(II) ions from the active site of urease. The urease inactivation by MIPs is comparable with that attained using NBPT. Urea hydrolysis in soil is accelerated 1015–fold by the nickel‐dependent enzyme urease to yield a source of N that can be assimilated by plants. This reaction determines an overall soil pH increase and significant ammonia volatilization, decreasing the efficiency of urea‐based fertilization. A control of urease activity is thus required for agronomic purposes. Maleic‐Itaconic Polymers (MIPs) have been claimed to decrease the N loss as ammonia volatilization by inhibiting urease activity. To investigate this matter, we performed an in vitro study using urease from jack bean (Canavalia ensiformis, JBU) at pH 7.5 and pH 5.0. Urease is not affected by MIPs at pH 7.5, but the enzyme is completely inactivated in 20 min at pH 5.0 using MIPs in the 0.4 to 2.4 μmol L–1 range. This inactivation is comparable to that attained using similar concentrations of N‐(n‐butyl)‐thiophosphoric triamide (NBPT). In vivo assays conducted using Sporosarcina pasteurii as a model for a widespread soil bacterium and urea, in the presence of either MIPs or NBPT at pH 7.5, revealed that NBPT significantly inhibits both cellular growth and urease activity, while MIPs have no effect. To elucidate the mechanism of extracellular urease inactivation by MIPs in vitro, their Ni(II) sequestration capability was investigated. The MIPs (50 μmol L–1) completely extract the essential Ni(II) ions from the active site of urease at pH 5.0, in 3 h. This phenomenon is related to the capability of MIPs to shift the Ni(II)‐urease dissociation equilibrium through binding and chelation of the Ni(II) ions off the active site.

Homotopy perturbation method for kinetic analysis of thermal inactivation of jack bean urease

In this work, theoretical modeling and determination of molar concentration of the native and denatured jack bean urease (EC 3.5.1.5) are presented. A three-reaction kinetic model of thermal inactivation of urease is analyzed using homotopy perturbation method. The obtained analytical solutions are used to study the kinetics of thermal inactivation of the enzyme as applied in biotechnology. From the results, it is established that the molar concentration of native enzyme decreases as the time increases while the molar concentration of the denatured enzyme increases as the time increases. The time taken to reach the maximum value of the molar concentration of native enzyme is the same as the time taken to reach the minimum value of the molar concentration of the denature enzyme. The molar concentration of the denatured enzyme reaches the steady state value when reaction time is less than or equal to 5s. Also, the molar concentration of the denatured enzyme becomes zero when rate constant of dissociation reaction of the native form of the enzyme into a denatured form, is less than or equal to 0.01 s−1. The analytical solutions are verified with numerical solutions using Runge–Kutta with shooting method and good agreements are established between the solutions. The information given in this theoretical investigation will assist in the kinetic analysis of the experimental results over handling rate constants and molar concentrations.

Urease catalytic behaviors induced by both urea and salt concentrations in ion-exchange hydrogels as dialysis membranes

This paper provides an insight into the urease catalytic responsive behaviors in ion-exchange hydrogels, subjected to variation of environmental concentrations of urea and sodium chloride with different urease enzymatic properties, including catalytic and Michaelis constants. Herein, a multiphysics model is formulated to quantify the catalytic activity of the immobilized urease, accounting for the multiphysics interaction between the environmental solution and the functional components of the urease-loaded charged hydrogel, including the immobilized urease, fixed charge group, the polymeric network chains. The effects of both the urease inactivation and denaturation are also incorporated into the model to capture the pH- coupled with temperature-induced urease catalytic behaviors. It is found that the urease catalytic activity patterns differ in anionic and cationic urease-loaded hydrogels, when subjected to the increase in environmental concentration of sodium chloride at a relatively higher environmental concentration of urea. It is also observed that the urease catalytic activity remains almost unchanged, when the environmental pH increases above the acid-base dissociation constant pK of the present charged hydrogel. Consequently, these findings would facilitate the development of a high-performance hydrogel-based dialysis membrane for highly efficient removal of the toxic urea and separation of the ionic metabolites in an wearable artificial kidney system.

The structure of urease inactivated by Ag(i): a new paradigm for enzyme inhibition by heavy metals

The nickel-dependent enzyme urease is a virulence factor for a large number of human pathogens, as well as a negative element for the efficiency of soil nitrogen fertilization for crop production. The use of urease inhibitors to contrast these effects requires the knowledge, at the molecular level, of their mode of action. Among these, silver is an efficient antimicrobial agent and an established inhibitor of this enzyme. The 1.91 Å resolution structure of Sporosarcina pasteurii urease inhibited by silver reveals the presence of two Ag(i) ions bound to the largely conserved triad αCys322/αHis323/αMet367: the first two residues are located on the mobile flap that is essential in modulating the size of the active site cavity and the position of key residues for enzyme catalysis, while αMet367 is on a loop facing the flap at the entrance of the active site cavity. The two Ag(i) ions are bridged by the thiolate Sγ atom of αCys322, and are coordinated, respectively, to the Nδ1 atom of the αHis323 imidazole ring and to the Sδ of αMet367. The binding of the Ag(i) ions at the edge of the active site channel supposedly blocks the movement of the flap, inhibiting the catalytic activity of urease. The structure of the silver-inhibited urease allows us to understand and rationalise all previously acquired kinetic and calorimetric data on this phenomenon, but also provides the details of how silver can exert its antimicrobial action with respect to ureolytic bacteria, a step forward against antibiotic-resistant pathogens.

PIF expression in endometriosis: Its possible role in inducing ectopic endometrium immune-privilege

In this paper, the dual urea- and pH-induced behaviors of the smart hydrogel in the biosensor system are examined at both the extremely low and high human physiological conditions. To the best of our knowledge, this is the first work to investigate the significant influence of the urea coupled with pH at both the extremes of physiological ranges on the responsive performance of the hydrogel, in which two correlations are integrated into the developed multiphysics model between the enzymatic reaction rate and diffusive hydrogen ionic concentration, and between the fixed charge group density and diffusive hydrogen ionic concentration. The effect of urease inactivation and denaturation due to environmental conditions are also incorporated into the model via a rate of reaction equation, including pH. The case studies are conducted with the focus on the responsive behavior of the hydrogels subject to both environmental pH and urea stimuli, simultaneously. The result reveals that the hydrogel may undergo the change of responsive characteristics from an anionic to a cationic hydrogel in response to the increase of the surrounding pH, subject to relatively higher urea cue such as 1000 mM. This work leads to a systematic study for rationally optimizing a urease-based medical device.

Characterization of dual urea- and pH-induced behaviors of smart hydrogel at human physiological conditions

In this paper, the dual urea- and pH-induced behaviors of the smart hydrogel in the biosensor system are examined at both the extremely low and high human physiological conditions. To the best of our knowledge, this is the first work to investigate the significant influence of the urea coupled with pH at both the extremes of physiological ranges on the responsive performance of the hydrogel, in which two correlations are integrated into the developed multiphysics model between the enzymatic reaction rate and diffusive hydrogen ionic concentration, and between the fixed charge group density and diffusive hydrogen ionic concentration. The effect of urease inactivation and denaturation due to environmental conditions are also incorporated into the model via a rate of reaction equation, including pH. The case studies are conducted with the focus on the responsive behavior of the hydrogels subject to both environmental pH and urea stimuli, simultaneously. The result reveals that the hydrogel may undergo the change of responsive characteristics from an anionic to a cationic hydrogel in response to the increase of the surrounding pH, subject to relatively higher urea cue such as 1000 mM. This work leads to a systematic study for rationally optimizing a urease-based medical device.

Inactivation of urease by catechol: Kinetics and structure

Urease is a Ni(II)-containing enzyme that catalyzes the hydrolysis of urea to yield ammonia and carbamate at a rate 1015 times higher than the uncatalyzed reaction. Urease is a virulence factor of several human pathogens, in addition to decreasing the efficiency of soil organic nitrogen fertilization. Therefore, efficient urease inhibitors are actively sought. In this study, we describe a molecular characterization of the interaction between urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) with catechol, a model polyphenol. In particular, catechol irreversibly inactivates both SPU and JBU with a complex radical-based autocatalytic multistep mechanism. The crystal structure of the SPU-catechol complex, determined at 1.50Å resolution, reveals the structural details of the enzyme inhibition.

Inactivation of urease by 1,4-benzoquinone: chemistry at the protein surface.

The high activity of urease, a Ni(ii) enzyme, has several adverse effects on human health and agriculture, and its modulation needs the use of inhibitors. 1,4-Benzoquinone (BQ) irreversibly inactivates Sporosarcina pasteurii urease (SPU), with first order kinetics for both the inhibitor and the enzyme. This reaction is stoichiometrically quenched in the presence of sulphite. The 2.07 Å crystal structure of SPU bound to BQ shows the presence of a 1,4-hydroquinone moiety covalently bound to the thiol group of αCys322, a key residue found on the mobile flap regulating the substrate access to the active site. The 1.75 Å crystal structure obtained when sulphite is added to a solution of SPU previously incubated with BQ shows the presence of a 2,5-dihydroxy-benzenesulphonate moiety bound to the αCys322 thiol group. These data reveal how the active site cysteine reacts with a prototypical BQ moiety, found in a large number of natural substances potentially suitable to control the urease activity.

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

BackgroundThe inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C.MethodsThe ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode.ResultsThe IC50 of ASB against JBU and HPU was 3.28 ± 0.13 mM and 3.17 ± 0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki = 2.86 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki* = 1.33 × 10−4 mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap.ConclusionsASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.