Ureases

The first enzyme that was demonstrated to contain nickel was urease (urea amidohydrolase) from jack bean. It catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The protein has a multimeric structure with a relative molecular mass of 590,000 Da. Analysis indicated 12 nickel atoms/mol. Binding studies with the inhibitors indicated an equivalent weight per active site of 105,000, corresponding to 2 nickel atoms/active site. During removal of the metal by treatment with EDTA at pH 3.7, the optical absorption and enzymatic activity correlated with nickel content. This, combined with the sensitivity of the enzyme to the chelating agents acetohydroxamic acid and phos-phoramidate, indicates that nickel is essential to the activity of the enzyme (1). 

The nickel in urease is nonmagnetic and appears to be in the oxidation state Ni(II). The broad optical absorption spectrum is influenced by ligands to the metal (Fig. 1). The spectrum obtained in the presence of the competitive inhibitor mercaptoethanol, after correction for Rayleigh scattering by the protein (31), shows absorption peaks at 324,380, and 420 nm, with molar absorption coefficients of 1550,890, and 460 A/-1 cm-1, respectively. These were assigned to sulfur-to-nickel charge transfer transitions. The spectrum is changed by addition of other inhibitors, such as acetohydroxamic acid (Fig. IB). Similar 

In extended X-ray absorption fine structure (EXAFS) studies of urease, Hasnain, Piggott, and co-workers (33, 34) demonstrated that spectra were similar to those of benzimidazole complexes, consistent 

Dixon et al. (35) have proposed a mechanism for urease catalysis (Fig. 3) based on studies of the reactions with the poor substrates formamide, acetamide, and iV-methylurea. They suggest that the two nickel ions are both in the active site, one binding urea and the other a hydroxide ion which acts as an efficient nucleophile. This implies that the nickel ions are within 0.6 nm (1 nm = 10 A) of each other so far it 

Biological catalysis by Ni can be divided in two broad classes the hydrolase activity represented by urease and the redox-associated mechanisms typical of hydrogenase, S-methyl coenzyme M reductases and CODHs. 

Ureases catalyse the hydrolysis of urea to ammonia and carbamate according to the reaction  

Hydrogenases catalyse the splitting or synthesis of the hydrogen molecule according to the reaction  

Finally, the one-electron reduction of the Ni-C form results in a new diamagnetic species called Ni-R. A plausible catalytic cycle can be postulated where the enzyme in the Ni-SI state binds H2 [81] yielding the two-electron more reduced Ni-R. Subsequent one-electron oxidations Ni-R — Ni-C — Ni-SI close the cycle. In addition to the redox changes, the various states differ in their degree of protonation, as indicated by the pH dependence of their redox potentials [87] (Fig. 12). 

Maroney and co-workers have argued that Ni is unlikely to be redox active under physiological conditions and that the redox process is most likely ligand-based [78]. Consequently, these authors have provided an alternative description of the paramagnetic Ni-C state as being generated by the interaction of a thyil radical with a Ni(II) ion. This species is isoelectronic with a Ni(III)-H bound to a thiolate according to the reaction  

Reactions of the same nature are observed with alanin, phenylglycin, and other similar substances. Althou the formation of aldehyde and the resulting liberation of ammonia is very slight and requires further confirmation, this decomposition is nevertheless interesting, for differing entirely from those thus far studied, it suggests a possible interpretation of the nature of the action of amidases, an interpretation which deserves further experimentation. 

The transformations which urine undergoes spontaneously have for a long time attracted the attention of investigators. Vauquelin and Dumas first recognized that the change of reaction found in urine exposed to the air corresponds to a transformation of urea into ammonium carbonate  

The urea ferments are widely distributed in nature, being found in the air, in the dunghill, in sewage and in dty mud. To cultivate these ferments, a solution of peptone with addition of 0.2-0.3 per cent of urea is used. According to Machida, the addition of magnesium sulphate to the culture medium greatly favors the development of bacteria. The fermentation of urea takes place especially well at a temperature of 30 at 5° almost no anunonia is formed at 60-70 , the cells of Uro-coccus succumb, though bacterial spores can be kept alive up to 80 . 

The catalyst intervening in the decoir sition of urea was discovered in 1874 by Musculus, who found that ammoniacal urine, filtered and evaporated in a vacuum, is capable of causing the fermentation of fresh urea, giving a thick and viscous product similar to that obtained by precipitating decomposed urine with alcohol. Musculus thus established that the production of ammonia is not due exclusively to the ferment, but claims that it results from the action of a special substance of enzymic nature secreted in the bladder. Thus, while finding the presence of the enzyme, Musculus did not comprehend the relation which exists between the bacterium and the active substance. It was Miquel who definitely demonstrated that the enzyme acting on urea is indeed secreted by a micro-organism. This special catalyst was first described under the name of urase, then under that of urease. 

To isolate urease, a solution of peptone is fermented by a culture of pure uro-bacteria with addition of from 2 to 3 g. of urea per liter the culture, kept at 30° becomes very turbid. After 3 or 4 days this is filtered through a porous cup, and the clear liquid, free from bacteria, can serve as a solution of urease. This liquor is very active. A liter can transform per hour as much as 120 g. of urea. Protected from the air, these solutions of urease can be preserved for months. The addition of two volumes of alcohol gives a precipitate which, washed in 

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Method, There are two standard methods for the estimation of urea, (i) the hypobromite method, (ii) the urease method (p. 519). The chief merit of the hypobromite method is the rapidity of the analysis the results obtained are considered sufficiently accurate for most medical requirements, e.g., for the estimation of urea in urine. For accurate metabolic work, however, the urease method should be employed. 

Urease soya bean, jack bean urea ammonium carbonate 7-2-7 9... 

The chief sources of this important enzyme are (a) the jack bean (Canavalia ensiformis). (b) the soy (or soja) bean (Glycine hispida). The enzyme is of great value in identifying and estimating urea. The action of urease on urea is specific, the reaction catalysed being ... 

Urease is one of the enzymes which have been obtained in the crystalline state. This has been done by stirring jack bean meal with 30°o aqueous acetone, filtering and allowing the filtrate to remain at o for several hours. The urease which crystallises out is separated by centrifuging and is then recrystallised. Like crystalline pepsin and trypsin, it is a protein. 

PRACTICAL ORGANIC CHEMISTRY ESTIMATION OF UREA BY UREASE. 

The method is based on the conversion of urea to amnionium carbonate and the estimation of the latter by titration with standard acid. For this purpose, two equal quantities of urea (or urine) are measured out into two flasks A and B. A is treated with 10 ml. of a strong urease preparation and some phenol-phthalein, warm water is added and the mixture is adjusted by the addition of V/io HCl from a burette A until the red colour is just discharged. This brings the mixture to about pH 8 (the optimum for urease) and also prevents loss of ammonia. 

The contents of B, which act as a control, are treated with mercuric chloride in order to inhibit the action of the enzyme, and then 10 ml. of urease solution are added. The solution is diluted with water and ammonium chloride added (in order to balance the ammonium chloride subsequently formed in A). Meth) l-red is then added and the solution is titrated with Mj 10 HCl from a second burette B until a bright red colour is obtained. 

Urease solution. Place about 5 g. of jack-bean meal in a mortar and grind up with about 10 ml. of water, t hen add about 90 ml. of water, mix thoroughly and allow to stand for some time in order to deposit starch and other insoluble substances. Decant off the supernatant liquid into a conical flask and cork the latter. 

The estimation. Label two 250 ml. conical flasks A and B, and into each measure 5 ml. of urine solution (or about o i g. of solid urea, accurately weighed). Add to each about 20 ml. of water and bring the temperature to about 60°. To A add 3 drops of phenolphthalein solution and to B add i ml. of 0-5% mercuric chloride solution. Now to each solution, add 10 ml. of the urease solution and mix well. The mixture A soon turns red. 

One example of an enzyme electrode is the urea electrode, which is based on the catalytic hydrolysis of urea by urease... 

In one version of the urea electrode, shown in Figure 11.16, an NH3 electrode is modified by adding a dialysis membrane that physically traps a pH 7.0 buffered solution of urease between the dialysis membrane and the gas-permeable... 

Schematic diagram of an enzyme-based potentiometric biosensor for urea in which urease is trapped between two membranes.

Directions are provided for constructing and characterizing an ammonium ion-selective electrode. The electrode is then modified to respond to urea by adding a few milligrams of urease and covering with a section of dialysis membrane. Directions for determining urea in serum also are provided. 

The enzyme urease catalyzes the hydrolysis of urea. The rate of this reaction was determined for a series of solutions in which the concentration of urea was changed while maintaining a fixed urease concentration of 5.0 pM. The following data were obtained. 

Reagent layer 1 porous gelatin coating film of urease and pH buffer... 

Arachin, the counterpart of glycinin in peanuts, consists of subunits of 60,000—70,000 mol wt which on reduction with 2-mercaptoethanol yield polypeptides of 41,000—48,000 and 21,000 mol wt (17) analogous to the behavior of glycinin. In addition to the storage proteins, oilseeds contain a variety of minor proteins, including trypsin inhibitors, hemagglutinins, and enzymes. Examples of the last are urease and Hpoxygenase in soybeans. 

Sodium hydrogen zirconium phosphate [34370-53-17 is an ion-exchange material used in portable kidney dialysis systems which regenerate and reckculate the dialysate solution. The solution picks up urea during the dialysis. The urea reacts with urease to form ammonia, which is absorbed by the sodium hydrogen zirconium phosphate. 

By the end of the nineteenth century a more descriptive system was in use. The suffix -ase was appended to the name of the substrate involved in the reaction, eg, amylase, ceUulase, protease, Hpase, urease, etc. Names that reflected the function of the enzyme with the suffix -ase were also used, eg, invertase, transferase, isomerase, oxidase. 

Hemodialysis with microencapsulated urease and an ammonia ion adsorbent, zirconium phosphate [13772-29-7], has been used (247) to delay the onset of dialysis therapy in patients retaining some renal function, and to reduce the time between dialysis treatment. 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Enzymes are proteins of high molecular weight and possess exceptionally high catalytic properties. These are important to plant and animal life processes. An enzyme, E, is a protein or protein-like substance with catalytic properties. A substrate, S, is the substance that is chemically transformed at an accelerated rate because of the action of the enzyme on it. Most enzymes are normally named in terms of the reactions they catalyze. In practice, a suffice -ase is added to the substrate on which die enzyme acts. Eor example, die enzyme dial catalyzes die decomposition of urea is urease, the enzyme dial acts on uric acid is uricase, and die enzyme present in die micro-organism dial converts glucose to gluconolactone is glucose oxidase. The diree major types of enzyme reaction are ... 

Bacterial catabolism of oral food residue is probably responsible for a higher [NHj] in the oral cavity than in the rest of the respiratory tract.Ammonia, the by-product of oral bacterial protein catabolism and subsequent ureolysis, desorbs from the fluid lining the oral cavity to the airstream.. Saliva, gingival crevicular fluids, and dental plaque supply urea to oral bacteria and may themselves be sites of bacterial NH3 production, based on the presence of urease in each of these materials.Consequently, oral cavity fNTi3)4 is controlled by factors that influence bacterial protein catabolism and ureolysis. Such factors may include the pH of the surface lining fluid, bacterial nutrient sources (food residue on teeth or on buccal surfaces), saliva production, saliva pH, and the effects of oral surface temperature on bacterial metabolism and wall blood flow. The role of teeth, as structures that facilitate bacterial colonization and food entrapment, in augmenting [NH3J4 is unknown. 

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