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)

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).
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PMID:Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site. 267 18

The genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3.5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.
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PMID:Regulation of gene expression and cellular localization of cloned Klebsiella aerogenes (K. pneumoniae) urease. 269 4

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.
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PMID:Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli. 283 33

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90,770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel-binding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.
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PMID:The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues. 340 46

Klebsiella aerogenes urease was purified 1,070-fold with a 25% yield by a simple procedure involving DEAE-Sepharose, phenyl-Sepharose, Mono Q, and Superose 6 chromatographies. The enzyme preparation was comprised of three polypeptides with estimated Mr = 72,000, 11,000, and 9,000 in a alpha 2 beta 4 gamma 4 quaternary structure. The three components remained associated during native gel electrophoresis, Mono Q chromatography, and Superose 6 chromatography despite the presence of thiols, glycols, detergents, and varied buffer conditions. The apparent compositional complexity of K. aerogenes urease contrasts with the simple well-characterized homohexameric structure for jack bean urease (Dixon, N. E., Hinds, J. A., Fihelly, A. K., Gazzola, C., Winzor, D. J., Blakeley, R. L., and Zerner, B. (1980) Can. J. Biochem. 58, 1323-1334); however, heteromeric subunit compositions were also observed for the enzymes from Proteus mirabilis, Sporosarcina ureae, and Selemonomas ruminantium. K. aerogenes urease exhibited a Km for urea of 2.8 +/- 0.6 mM and a Vmax of 2,800 +/- 200 mumol of urea min-1 mg-1 at 37 degrees C in 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid, 5.0 mM EDTA buffer, pH 7.75. The enzyme activity was stable in 1% sodium dodecyl sulfate, 5% Triton X-100, 1 M KCl, and over a pH range from 5 to 10.5, with maximum activity observed at pH 7.75. Two active site groups were defined by their pKa values of 6.55 and 8.85. The amino acid composition of K. aerogenes urease more closely resembled that for the enzyme from Brevibacter ammoniagenes (Nakano, H., Takenishi, S., and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495-1502) than those for plant ureases. Atomic absorption analysis was used to establish the presence of 2.1 +/- 0.3 mol of nickel per mol of 72,000-dalton subunit in K. aerogenes urease.
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PMID:Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. 355 84

Sixty-four Angus steers initially averaging 354 kg were allotted to a 2 X 2 factorial arrangement of treatments to determine the effects of dietary Ni (0 or 5 mg/kg supplemental), monensin (0 or 33 mg/kg) and their possible interaction on performance, methane production and N metabolism. The basal diet was a high energy, corn-cottonseed hull based diet containing 10.2% crude protein and .30 mg/kg Ni on a dry matter basis. Monensin reduced (P less than .05) feed intake, did not affect average daily gain and improved (P less than .05) feed conversion over the 102-d study. Nickel supplementation did not significantly alter or interact with monensin to affect steer performance. However, steers fed Ni tended to have higher average daily gains and improved feed conversions. Monensin decreased (P less than .05) in vitro methane production, altered several carcass traits, increased (P less than .05) molar proportion of ruminal propionate and decreased (P less than .05) molar proportion of ruminal acetate. Nickel did not alter methane production, carcass characteristics or ruminal volatile fatty acid proportions. Both monensin and Ni increased (P less than .05) ruminal fluid urease activity when samples were obtained before feeding. A significant monensin X Ni interaction was found to affect ruminal epithelial urease activity. Monensin increased ruminal epithelial urease in steers not receiving supplemental Ni, but had no effect on ruminal epithelial urease activity in steers fed supplemental Ni. Ruminal fluid protein and ammonia-N were decreased (P less than .05) by monensin. Results of this study indicate that Ni may interact with monensin to affect ruminal epithelial urease activity but not performance or methane production in finishing steers.
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PMID:Performance, methanogenesis and nitrogen metabolism of finishing steers fed monensin and nickel. 357 Oct 11

Urease was purified 592-fold to homogeneity from the anaerobic rumen bacterium Selenomonas ruminantium. The urease isolation procedure included a heat step and ion-exchange, hydrophobic, gel filtration, and fast protein liquid chromatography. The purified enzyme exhibited a Km for urea of 2.2 +/- 0.5 mM and a Vmax of 1100 mumol of urea min-1 mg-1. The molecular mass estimated for the native enzyme was 360,000 +/- 50,000 daltons, whereas a subunit value of 70,000 +/- 2,000 daltons was determined. These results are in contrast to the findings of Mahadevan et al. (Mahadevan, S., Sauer, F. D., and Erfle, J. D. (1977) Biochem. J. 163, 495-501) in which isolated rumen urease was reported to be one-third this size (Mr 120,000-130,000) and to catalyze urea hydrolysis at a maximum velocity of only 53 mumol min-1 mg-1. S. ruminantium urease contained 2.1 +/- 0.4 nickel ions/subunit, comparable to the nickel content in jack bean urease (Dixon, N.E., Gazzola, C., Blakeley, R.L., and Zerner, B. (1975) J. Am. Chem. Soc. 97, 4131-4133). Thus, the active site of bacterial urease is very similar to that found in the plant enzymes.
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PMID:Purification of a nickel-containing urease from the rumen anaerobe Selenomonas ruminantium. 371 Nov 13

Urease from Bacillus pasteurii DSM 33 was purified 34-fold to a maximum specific activity of 996.5 mumol urea min-1 mg-1 at 30 degrees C. Homogeneity was demonstrated by isoelectric focussing which showed a single protein zone corresponding to a pI of about 4.6. The native enzyme was demonstrated to have a molecular mass of 230,000 and to consist of identical subunits of 65,500, as measured by SDS electrophoresis. Radioactive 63Ni-nickel co-chromatographed with urease through gel filtration, ion-exchange, and affinity chromatography. Measuring specific radioactivity, the nickel content was found to be 1.00 (+/- 0.1) g-atom Ni per mol of subunit, and 0.82 g-atom Ni per mol as measured by atomic absorption spectrometry. This indicates that 1 atom of nickel is present in each of four subunits of the enzyme.
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PMID:Nickel-content of urease from Bacillus pasteurii. 375 42

Thirty male calves were used in a 2 X 3 factorial arrangement of treatments to determine the effects of dietary nickel and protein on performance, urease activity and tissue concentrations of nickel, iron, zinc, copper and manganese. Protein levels evaluated were 10.0, 12.25 and 14.5%, and nickel was supplemented at a level of 0 or 5 mg/kg of diet. Nickel did not affect growth during the 140-d study but tended to increase efficiency of gain in calves fed 14.5% protein. Rumen fluid urease activity was increased by nickel only in animals receiving the low protein diet. Urease activity in rumen fluid was higher in calves fed 10.0% than in animals fed 12.25% or 14.5% protein. Neither nickel nor protein affected urease activity in rumen epithelium. Increasing dietary protein resulted in increased urease in cecal digesta. Lung, liver, kidney and serum nickel concentrations were increased by supplemental nickel. A nickel X protein interaction was noted for kidney nickel. Nickel supplementation increased kidney nickel to a greater degree in calves fed 10.0% protein than in calves fed higher protein levels. Nickel supplementation reduced iron concentrations in lung, liver and muscle and manganese concentrations in muscle. Increased dietary protein decreased iron in liver and spleen but increased manganese concentrations in heart. These findings indicate that dietary protein influences responses of ruminants to nickel supplementation and relatively small increases in dietary nickel and protein can influence metabolism of other trace elements.
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PMID:Effects of dietary nickel and protein on growth, nitrogen metabolism and tissue concentrations of nickel, iron, zinc, manganese and copper in calves. 377 17

Evidence is reviewed that indicates Ni is an essential element for the chick, rat, pig, sheep and goat. Although a number of possible functions for Ni have been proposed based on in vitro and in vivo studies, the physiological role of Ni in the mammalian or avian system is presently unknown. Rumen bacterial urease has been shown to be a Ni-dependent enzyme and Ni is a component of factor F430 present in methanogenic bacteria. Nickel can interact or influence the metabolism of a number of minerals. Interactions of Ni and Fe, Zn and Cu are discussed. The requirement for Ni is low (50 to 60 ppb) in chicks fed semipurified diets. Insufficient data are available to estimate the Ni requirement of swine. In ruminants, the Ni requirement appears to be higher than that for other animal species. Nickel supplementation to practical diets has increased gain, feed efficiency and ruminal urease activity in ruminants, but performance results have been inconsistent. Level of crude protein and urea are two factors that influence ruminant responses to dietary Ni. The greatest responses have been observed in ruminants fed low protein diets. Little is known concerning levels, forms and bioavailability of Ni in different feedstuffs. Nickel is homeostatically controlled in the animal's body and high levels of Ni are required to cause toxicity.
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PMID:Nickel as a "newer trace element" in the nutrition of domestic animals. 638 82


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