Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.4.6 (urease)
 7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively.
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PMID:Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids. 329 57

The influence of four inhibitors: boric acid, thioglycolic acid, sodium fluoride and acetohydroxamic acid on the activity of urease, both in the native form and immobilized covalently on glutaraldehyde-pretreated chitosan membrane, was studied. Urea hydrolysis was carried out in phosphate buffer, pH 7, at 25 degrees C at urea concentration of 50 mM. The immobilized urease was more resistant than the native one to the action of all the investigated inhibitors, except boric acid. This property of the enzyme offers a possibility of its practical application.
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PMID:Competitive inhibitors of free and chitosan-immobilized urease. 765 53

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.
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PMID:Diethylpyrocarbonate reactivity of Klebsiella aerogenes urease: effect of pH and active site ligands on the rate of inactivation. 842 33

The urease from the picoplanktonic oceanic Prochlorococcus marinus sp. strain PCC 9511 was purified 900-fold to a specific activity of 94.6 micromol urea min(-1) (mg protein)(-1) by heat treatment and liquid chromatography methods. The enzyme, with a molecular mass of 168 kDa as determined by gel filtration, is the smallest urease known to date. Three different subunits with apparent molecular masses of 11 kDa (gamma or UreA; predicted molecular mass 11 kDa), 13 kDa (ss or UreB; predicted molecular mass 12 kDa) and 63 kDa (alpha or UreC; predicted molecular mass 62 kDa) were detected in the native enzyme, suggesting a quaternary structure of (alphassgamma)(2). The K:(m) of the purified enzyme was determined as being 0.23 mM urea. The urease activity was inhibited by HgCl(2), acetohydroxamic acid and EDTA but neither by boric acid nor by L-methionine-DL-sulfoximine. Degenerate primers were designed to amplify a conserved region of the ureC gene. The amplification product was then used as a probe to clone a 5.7 kbp fragment of the P. marinus sp. strain PCC 9511 genome. The nucleotide sequence of this DNA fragment revealed two divergently orientated gene clusters, ureDABC and ureEFG, encoding the urease subunits, UreA, UreB and UreC, and the urease accessory molecules UreD, UreE, UreF and UreG. A putative NtcA-binding site was found upstream from ureEFG, indicating that this gene cluster might be under nitrogen control.
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PMID:Prochlorococcus marinus strain PCC 9511, a picoplanktonic cyanobacterium, synthesizes the smallest urease. 1110 68

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.
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PMID:Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis. 1186 12

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.
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PMID:Irreversible inhibition of jack bean urease by pyrocatechol. 1469 8

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).
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PMID:Inactivation of jack bean urease by allicin. 1469 9

The structure of the complex of urease, a Ni-containing metalloenzyme, with boric acid was determined at 2.10 A resolution. The complex shows the unprecedented binding mode of the competitive inhibitor to the dinuclear metal center, with the B(OH)3 molecule bridging the Ni ions and leaving in place the bridging hydroxide. Boric acid can be considered a substrate analogue of urea, and the structure supports the proposal that the Ni-bridging hydroxide acts as the nucleophile in the enzymatic process of urea hydrolysis.
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PMID:Molecular details of urease inhibition by boric acid: insights into the catalytic mechanism. 1503 15

In this work, an analytical system based on the coupling of gas diffusion separation and sequential injection analysis for urea determination in milk is presented. A versatile manifold that could simultaneously be used for either spectrophotometric or conductometric detection was constructed. The sample and urease solution are sequentially aspirated into the holding coil and sent to a thermoreactor, where urea is enzymatically hydrolyzed by urease and converted into ammonium. This stream merges an alkaline solution at a confluence point where ammonia is formed. Ammonia diffuses through a hydrophobic membrane and modifies the bromothymol blue indicator color, when spectrophotometric detection is used, or changes the conductance of a boric acid solution acceptor stream, when conductometric detection is used. This methodology was applied to the determination of urea in 18 milk samples and the results were statistically comparable with those furnished by the enzymatic recommended procedure. The detection limits were 2.6 x 10(-4) and 2.8 x 10(-5) mol L(-1) for conductometric and spectrophotometric detection, respectively. Repeatability (relative standard deviation, RSD) was better than 3.7% and 2.6% for conductometric and spectrophotometric detection, respectively.
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PMID:Enzymatic determination of urea in milk by sequential injection with spectrophotometric and conductometric detection. 1553 91

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 pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(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 pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(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 pK(a)=6.6 is a merger of two pK(a)s 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.
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PMID:Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation. 1602 57


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