Active Site Of Urease Research Articles

A model‐based proposal for the role of UreF as a GTPase‐activating protein in the urease active site biosynthesis

UreF is a protein that plays a role in the in vivo urease activation as a chaperone involved in the insertion of two Ni(2+) ions in the apo-urease active site. The molecular details of this process are unknown. In the absence of any molecular information on the UreF protein class, and as a step toward the comprehension of the relationships between UreF function and structure, we applied a structural modeling approach to infer useful biochemical knowledge on Bacillus pasteurii UreF (BpUreF). Similarity searches and multiple alignment of UreF protein sequences indicated that this class of proteins has a low homology with proteins of known structure. Fold recognition methods were therefore used to identify useful protein structural templates to model the structure of BpUreF. In particular, the templates belong to the class of GTPase-activating proteins. Modeling of BpUreF based on these templates was performed using the program MODELLER. The structure validation yielded good statistics, indicating that the model is plausible. This result suggests a role for UreF in urease active site biosynthesis as a regulator of the activity of UreG, a small G protein involved in the in vivo apo-urease activation process and established to catalyze GTP hydrolysis.

Biochemical Studies on Mycobacterium tuberculosis UreG and Comparative Modeling Reveal Structural and Functional Conservation among the Bacterial UreG Family

Nickel is a fundamental micronutrient for cellular life, but it is toxic in soluble form at nonphysiological concentrations. Such potentially contradictory features required living organisms to develop efficient systems for nickel utilization and homeostasis. This is the case for incorporation of nickel into the active site of urease, a multistep, tightly regulated process, requiring the interplay of various accessory proteins. The understanding of this activation mechanism may find medical applications against ureolytic bacteria, among which Mycobacterium tuberculosis is a deadly pathogen for humans. The topic of this study is UreG, an essential chaperone in the in vivo activation of urease upon insertion of Ni2+ into the active site. The protein was examined using both experimental and computational approaches. In particular, the soluble M. tuberculosis UreG (MtUreG) was overexpressed in Escherichia coli and purified to homogeneity. The identity of the isolated protein was established by mass spectrometry. On-line size-exclusion chromatography and light scattering indicated that MtUreG exists as a dimeric form in solution. Determination of the free thiol concentration revealed that a disulfide bond is present in the dimer. The isolated MtUreG shows low GTPase activity under native conditions, with a kcat of 0.01 min-1. Circular dichroism spectroscopy demonstrated the presence of a well-defined secondary structure (8% alpha-helices, 29% beta-strands) in MtUreG, whereas NMR spectroscopy indicated that this protein does not behave as a rigid three-dimensional fold and thus can be assigned to the class of intrinsically unstructured polypeptides. The molecular model of MtUreG in the fully folded and functional form was built using fold recognition algorithms. An extensive similarity search was performed to determine conservation patterns in all known bacterial UreG sequences. The generation of a multiple-sequence alignment and the related phylogenetic tree allowed us to recognize key residues and motifs that are likely important for protein function. A structural database containing the homology-built models of the most representative UreG proteins was created, confirming the structural analogies among the UreG family. A flexible region, likely to be important for protein function, is identified. The structural conservation among this class of GTPases is discussed on the basis of their function in the urease assembly process.

Geometry optimization method based on approximate spin projection and its application to F2, CH2, CH2OO, and active site of urease

AbstractA new geometry optimization method based on an approximate spin projection (AP) procedure is proposed to eliminate a spin contamination effect in an optimized structure on a low spin (LS) state of a broken symmetry (BS) method. First, an energy gradient with the AP (AP gradient) is derived and it is applied to the geometry optimization of F2, CH2, and CH2OO in order to obtain their structures without the spin contamination. The optimization method corrects HCH angle of the CH2 about 10° in comparison with the BS method, and indicates that the spin contamination in optimized geometry of the BS LS states is considerably large. Next, an active site of Urease that consists of two Ni(II) ions is optimized by the AP method. The result indicates that structure of the active site is sensitive to a water molecule called W3. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

The Nickel Site of Bacillus pasteurii UreE, a Urease Metallo-Chaperone, As Revealed by Metal-Binding Studies and X-ray Absorption Spectroscopy

UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of Ki* = 4.5 × 10− 7 mM. The respective inhibition constant of TCoBQ was equal to: Ki* = 2.4 × 10− 6 mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.

μ-Hydroxo-μ-phenolato Dinuclear Nickel(II) Complexes in a Mixed-Spin State and Their Urea Adduct as Relevance to the Urease Active Site

Abstract Dinucleating compartmental ligands (2,6-bis{N-[(2-dimethylamino)ethyl]iminomethyl}-4-methylphenol (HL1) and 2-{N-[(2-dimethylamino)ethyl]iminomethyl}-6-{N-methyl-N-[2-(dimethylamino)ethyl]aminomethyl}-4-methylphenol (HL2)) have afforded the following dinuclear nickel(II) complexes: [Ni2(L1)(OH)(py)(H2O)](ClO4)2·H2O (1), [Ni2(L2)(OH)(MeOH)(H2O)](ClO4)2·CH3CN (2), and [Ni2(L2)(OH)(dmf)2](ClO4)2 (3). X-ray crystallographic studies for 1 and 3 have clarified a μ-hydroxo-μ-phenolato dinuclear structure in a mixed-spin state with one pseudo octahedral Ni(II) (S = 1) and one planar Ni(II) (S = 0). The urea adducts, [Ni2(L1)(OH)(urea)(H2O)](ClO4)2·H2O (1′) and [Ni2(L2)(OH)(urea)(H2O)](ClO4)2 (2′), were prepared by the reaction of 1 and 2 with urea, respectively, and were structurally characterized to be in the mixed-spin state. The relevance of 1′ and 2′ to urease is discussed.

Who's the Most Proficient of All?

BIOCHEMISTRY It might seem a straight-forward matter to determine how much the enzyme urease accelerates the rate of decomposition of urea, which, after all, contains only four nonhydrogen atoms. The problem, however, is that in the nonenzymatic pathway, the elimination of ammonia precedes the addition of water, whereas the enzyme promotes the nucleophilic attack of water, yielding a tetrahedral intermediate and a different reaction pathway to the same products. Previously, Estiu and Merz carried out a computational analysis of the uncatalyzed reaction, relying both on earlier studies with small dinickel molecules and on structural analysis of the dinickel cluster at the urease active site. They found that the catalytic proficiency of the enzyme, calculated by dividing the biochemical quantity k cat/ K m by the rate constant of the noncatalyzed reaction ( k non), is many orders of magnitude greater than that of the reigning champion, ornithine 5'-monophosphate decarboxylase. In contrast, Callahan et al. have measured the hydrolysis of substituted ureas, which cannot undergo elimination, and extrapolated from these data to arrive at a much faster non-catalyzed hydrolysis rate—one that confirms urease as a proficient, but not record-setting, catalyst. — GJC J. Am. Chem. Soc. 126 , 6932; 10.1021/ja0525399 (2005).

The Burden Borne by Urease

At the active site of urease, urea undergoes nucleophilic attack by water, whereas urea decomposes in solution by elimination of ammonia so that its rate of spontaneous hydrolysis is unknown. Quantum mechanical simulations have been interpreted as indicating that urea hydrolysis is extremely slow, compared with other biological reactions proceeding spontaneously, and that urease surpasses all other enzymes in its power to enhance the rate of a reaction. We tested that possibility experimentally by examining the hydrolysis of 1,1,3,3-tetramethylurea, from which elimination cannot occur. In neutral solution at 25 degrees C, the rate constant for the uncatalyzed hydrolysis of tetramethylurea is 4.2 x 10-12 s-1, which does not differ greatly from the rate constants observed for the uncatalyzed hydrolysis of acetamide (5.1 x 10-11 s-1) or N,N-dimethylacetamide (1.8 x 10-11 s-1) under the same conditions. We estimate that the proficiency of urease as a catalyst, (kcat/Km)/knon, is 8 x 1017 M-1, slightly higher than the values for other metalloenzymes (carboxypeptidase b and cytidine deaminase) that catalyze the hydrolysis of similar bonds.

Theoretical studies on magnetic interactions between Ni(II) ions in urease

Abstract The chemical species of bridge oxygen (WB) in active site of urease was examined in terms of a magnetic interaction between Ni(II) ions. The effective exchange integrals (Jab) values between Ni(II) ions were calculated by using three candidates of WB, i.e., H2O, OH− and O2−. The unrestricted hybrid density functional theory (UHDFT) calculations were carried out using the smallest models and realistic models. In the case of the smallest models, the experimental Jab value (−6.3 cm−1) was a middle value of WB = H2O (−2.6 cm−1) and WB = OH− (−11.4 cm−1), while we could not obtain appropriate electronic structure using O2−-bridge model. The Jab became −16.7 cm−1 by using more realistic OH−-bridge model, while one spin density on Ni2 ion was delocalized to Asp360 in case of H2O-bridge model. Natural orbital analyses revealed that the magnetic orbital through Lys-bridge delocalized to Asp360 and it seemed to associate with the spin transfer. From the results, OH−-bridge seemed to be the most possible candidate for the structure of active site in urease.

Jack bean ( Canavalia ensiformis) urease. Probing acid–base groups of the active site by pH variation

A pH-variation study of jack bean ( Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with p K as equal to 5.3 ± 0.2, 6.6 ± 0.2 and 9.1 ± 0.4. Based on the appearance of the ionization step with p K a = 5.3 in v max-pH, K M-pH and K i-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v max-pH and K M-pH curves, the obvious role of the group with p K a = 9.1 is the participation in the catalytic reaction. One function of the group featuring p K a = 6.6, which was derived from a two-maxima v max-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease–phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the p K a = 6.6 is a merger of two p K as close in value. The study establishes that regular bell-shaped activity–pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.

UreG, a Chaperone in the Urease Assembly Process, Is an Intrinsically Unstructured GTPase That Specifically Binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.

Enzymatic catalysis of urea decomposition: elimination or hydrolysis?

We present a high-level quantum chemical study of possible reaction mechanisms associated with the catalytic decomposition of urea by a bioinorganic mimetic of the dinickel active site of urease. We chose the phthalazine-dinickel complexes of Lippard and co-workers, because these mimetics have been shown to hydrolytically degrade urea. High-level quantum chemical methodologies were utilized to identify stable intermediates and transition-state structures along several possible reaction pathways. The computed results were then used to further analyze what may occur in the active site of urease. Valuable information on the latter has been extracted from experimental data, computational approaches, and unpublished molecular dynamics simulations. On the basis of these comparative studies, we propose that both the elimination and hydrolytic pathways may compete for urea decomposition in the active site of urease.

The Hydrolysis of Urea and the Proficiency of Urease

We present the results of a computational study of the solution phase decomposition of urea, which provides insight into probable reaction pathways for the urease-catalyzed reaction. Calculations, which were used to derive thermodynamic parameters that were further used for a kinetic analysis, have been done at the solvent-corrected MP2/6-311++G** level. Both elimination and hydrolytic pathways have been considered. Elimination is favored for the solution phase reaction, which proceeds by H-bond coordination of a water molecule to the amine nitrogen atoms. The coordination of one water molecule greatly facilitates the reaction by allowing it to proceed through a cyclic six-member transition state. Aspects of the water-urea H-bond interactions have also provided insights into critical aspects of the hydrogen bond pattern in the urease active site. On the basis of a kinetic analysis, we have estimated the proficiency of urease and have predicted that it is the most proficient enzyme identified to date.

Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone

UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni2+ ions in the active site of urease. This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking. The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed. This analysis provided insights into the protein structural and conformational features. A structural database of UreE proteins from a large number of different genomes was built using homology modeling. All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE. A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures. The quality of all models was tested using standard assessment procedures. The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly.

A study of the inhibition of jack bean urease by garlic extract

An inhibiting effect of fresh garlic extract (GE) on jack bean urease has been investigated in order to elucidate the kinetics and mechanism of inhibition. The system: jack bean urease-garlic extract-urea, can be treated as a model system in the studies on the utility of garlic in the therapy of diseases caused by Helicobacter pylori and other bacteria producing urease. The influence of fresh aqueous garlic extract on the activity of jack bean urease at 22 °C in 20 mM phosphate buffer, pH 7 was investigated. The loss of urease activity was found to be directly proportional to alk(en)yl thiosulfinates (TS) content in the garlic extract. The inhibitory activity of GE was compared with the activity of synthetic allicin. The results indicate that the inhibition of urease by GE is irreversible and incubation time-dependent. The kinetics of the inhibition was found to be biphasic; each phase obeyed first-order kinetics. Thiol reagents (L-cysteine, 2-mercaptoethanol, glutathione, dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while urea and boric acid show weaker protection. GE-modified urease could be reactivated with dithiothreitol. This study demonstrates, that the inhibition of urease by GE should be atrributed to the reaction of TS with the SH-group (Cys 592) found in the active site of urease.

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).

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°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.

Tunable TACN/pyrazolate hybrid ligands as dinucleating scaffolds for metallobiosite modeling—dinickel(II) complexes relevant to the urease active site

Two new dinucleating pyrazole ligands HL1 and HL2 bearing appended 1,4-diisopropyl-1,4,7-triazacyclononane (iPr2TACN) side arms in the 3- and 5-positions have been synthesized by multi-step synthetic procedures. HL1 and HL2 differ by the length of the spacer between the pyrazole and the iPr2TACN, which is a novel means of tuning the metal–metal-separation in pyrazolate-based bimetallic compounds. Complexes [L1Ni2(H3O2)](ClO4)2 (5) and [L2Ni2(OH)](BPh4)2 (6b) have been characterized structurally, where the bridging H3O2 moiety in 5 is reactive and allows cooperative substrate transformations within the bimetallic pocket, while the tightly bound OH bridge in 6b is inert. Using the dinickel scaffold 5, reactivity relevant to the urease active site has been probed. Under anhydrous conditions, 5 reacts with parent and N-substituted urea to yield complexes 8a–c featuring N,O-bridging ureate anions. At high temperatures, 8a–c lose ammonia to give the cyanate-bridged species 10. This is able to bind a second cyanate at one of the nickel ions forming 11. Complexes with bridging acetate (7), O,O-bridging carbamate (9) and N,O-bridging methyl carbamate (12) have been studied for comparison, and mutual interconversions of the dinickel complexes have been investigated. Complexes 5, 6b, 7, 8a–c, 9, 11 and 12 have been characterized by X-ray crystallography.

Effects of salt and nickel ion on the conformational stability of Bacillus pasteurii UreE

UreE, a urease accessory protein, is proposed to be a metallochaperone assisting the nickel incorporation into the urease active site. We investigated the effects of salt and nickel on the conformational stability of the UreE from Bacillus pasteurii ( BpUreE), by circular dichroism (CD) and nuclear magnetic resonance spectroscopy accompanying a thermodynamic inspection. Far-UV CD spectra of BpUreE showed that both salt and nickel stabilized the ordered structure of the protein. The thermal denaturing of BpUreE showed a bimodal feature with an aggregation process before thermal unfolding. This thermally induced aggregation could be suppressed by the addition of salt up to 50 mM, and the further addition of salt increased the thermal resistance of the protein. The nickel addition also elevated the thermal resistance of BpUreE, although it could not prevent the aggregating process. Additionally, the stoichiometry of a specific nickel binding to BpUreE was revealed as one nickel per dimer. Altogether, the present results establish a rather detailed characterization of the thermostability and nickel-binding property of BpUreE.