Urease from Arthrobacter oxydans,a nickel-containing enzyme

In Arthrobacter oxydans, Klebsiella aerogenes and Sporosarcina ureae, growth with urea as a nitrogen source turned out to be more sensitive to inhibition by EDTA than that with ammonia. The inhibition was overcome by added nickel chloride, but not by other divalent metal ions tested. In A. oxydans the uptake of ⁶³Ni was paralleled by an increase in urease (urea amidohydrolase, EC 3.5.1.5) activity under certain conditions. Following growth with radioactive nickel, urease from this strain was enriched by heat treatment and acetone fractionation. Copurification of ⁶³Ni and urease was observed during subsequent Sephadex gel chromatography. Almost the entire labelling was detected together with the purified enzyme after focusing on polyacrylamide gel. The relative molecular mass of the purified urease was estimated to be 242,000. The pH optimum was 7.6, the K _m-value 12.5 mmol/l and the temperature optimum 40°C; heat stability was observed up to 65°C. In presence of 10 mmol/l EDTA the protein-nickel binding remained intact at pH 7; at pH 5 and below, nickel was irreversibly removed with concommitant loss of enzyme activity. The results demonstrated that nickel ions are required for active urease formation in the bacterial strains studied, and that urease from A. oxydans is a nickel-containing enzyme.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Inactivation of jack bean urease by allicin

Allicin--diallyl thiosulfinate--is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20 mM phosphate buffer, pH 7.0 at 22 degrees C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).     

Inactivation of Jack Bean Urease by Allicin

Allicin—diallyl thiosulfinate—is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20?mM phosphate buffer, pH 7.0 at 22°C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).     

Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme

The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activity was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg (2+) and Hg2(2+) inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2+ ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2+ binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2+, whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence, reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 degrees C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation

Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site

We examined several compounds for their mechanisms of inhibition with the nickel-containing active site of homogeneous Klebsiella aerogenes urease. Thiolate anions competitively inhibit urease and directly interact with the metallocenter, as shown by the pH dependence of inhibition and by UV-visible absorbance spectroscopic studies. Cysteamine, which possesses a cationic beta-amino group, exhibited a high affinity for urease (Ki = 5 microM), whereas thiolates containing anionic carboxyl groups were uniformly poor inhibitors. Phosphate monoanion competitively inhibits a protonated form of urease with a pKa of less than 5. Both the thiolate and phosphate inhibition results are consistent with charge repulsion by an anionic group in the urease active site. Acetohydroxamic acid (AHA) was shown to be a slow-binding competitive inhibitor of urease. This compound forms an initial E.AHA complex which then undergoes a slow transformation to yield an E.AHA* complex; the overall dissociation constant of AHA is 2.6 microM. Phenylphosphorodiamidate, also shown to be a slow-binding competitive inhibitor, possesses an overall dissociation constant of 94 pM. The tight binding of phenylphosphorodiamidate was exploited to demonstrate the presence of two active sites per enzyme molecule. Urease contains 4 mol of nickel/mol enzyme, hence there are two nickel ions/catalytic unit. Each of the two slow-binding inhibitors are proposed to form complexes in which the inhibitor bridges the two active site nickel ions. The inhibition results obtained for K. aerogenes urease are compared with inhibition studies of other ureases and are interpreted in terms of a model for catalysis proposed for the jack bean enzyme (Dixon, N.E., Riddles, P.W., Gazzola, C., Blakely, R.L., and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344).     

The metal dependence of Bacillus subtilis phytase

The metal ion requirement of a Bacillus subtilis phytase has been studied. Removal of metal ions from the enzyme by EDTA resulted in complete inactivation. Circular dichroism spectroscopy was used to study the effect of metal ion removal on the protein conformation. The loss of enzymatic activity is most likely due to a conformational change, as the circular dichroism spectra of holoenzyme and metal-depleted enzyme were different. Metal-depleted enzyme was partially able to restore the active conformation when incubated in the presence of calcium. Only minor reactivation was detected with other divalent metal ions and their combinations. Based on the data we conclude that B. subtilis phytase requires calcium for active conformation. Calcium has also a strong stabilizing effect on the enzyme against thermal denaturation. However, the conformational change resulted by calcium depletion does not affect the protease susceptibility.     

Characteristics of Ureaplasma urealyticum urease.

Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins. The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease. The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate. Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid. Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Barrier function of the cell wall during uptake of nickel ions

Cell walls were isolated from roots of six plant species to study their ion-exchange capacity for nickel ions (S _Ni) at Ni²⁺ concentration of 10⁻³ M. The S _Ni values varied depending on the plant species from 50 to 150 μmol Ni²⁺ per gram dry wt; the sorption capacity increased in a row: Poaceae < Chenopodiaceae < Fabaceae. At pH 5 the sorption capacity of cell walls for nickel ions was determined by the presence of carboxyl groups of polygalacturonic acid in the polymeric cell-wall matrix. In all cases the ion-exchange capacity of cell walls was higher at pH 8 than at pH 5, indicating that Ni²⁺ binds also to a carboxyl group different from that of polygalacturonic acid. Irrespective of plant species, the presence of EDTA in the solution diminished drastically the absorption capacity of cell walls for Ni²⁺. It is concluded that the presence of 10⁻³ M EDTA weakens the defense properties of cell walls. The sequestration of Ni²⁺ in the cell wall can be considered as an effective means of plant cell defense against elevated concentrations of nickel ions in the external medium.     

Structural and functional role of nickel ions in urease by molecular dynamics simulation

Nickel ions play several roles in the biological processes of microorganisms and plants. Urease has a nickel-containing active site and catalyzes the hydrolysis of urea to yield ammonia and carbamate. In the present study, the role of nickel ions is examined using molecular dynamics simulations of the holo and apo forms. Nonbonded models used for the nickel ions provide good reproduction of the active-site structure as indicated in the crystallized structure. The results confirm that urease has a rigid active site in either its holo or its apo form. A new conformation of the flap is observed in apo urease. The connection between the metal center and His^α323 is proposed to be responsible for maintaining the flap conformation. The binding free energy of acetohydroxamic acid and urease is estimated using the molecular mechanics–generalized Born/surface area method. The binding free energy is primarily driven by electrostatic interactions in the presence of nickel ions. Normal mode analysis is employed to characterize the movements of the flap in apo urease.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

Irreversible inhibition of jack bean urease by pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 25 degrees C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as L-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.