Urease substrates

Urease is enzymatically active over a broad pH range. The specificity of urease for its substrate (urea) is almost absolute. The urease substrate solution contains urea and a pH indicator, bromocresol purple, at pH 4.7. The urease eatalyzes the urea into ammonia and bicarbonate, and the released ammonia causes an increase in pH that changes the color of the indieator from yellow to purple. The generation of color is not directly related to the amount of urea catalyzed. Because the color development is dependent on pH, it is essential to cheek that the pH is accurate before addition to wells in a test. It is also essential that no alkaline buffers remain after, e.g., washing (pH 7.4, PBS) because this will cause a change in color, and plates must be washed finally in water if PBS is the usual washing buffer. 

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. 

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 ... 

Most enzymes are quite specific for a given substrate. For example, the enzyme urease that catalyzes the reaction... 

Urea Electrodes The physiologically important substrate urea can be sensed based on the following urease-catalyzed reaction ... 

Enzymes can perform a multitude of readions, though every enzyme usually only catalyzes very specifically the readion of a single substrate. They are named after the readion they catalyze, or the substrate with which they read, by adding the suffix -ase. Hence there are oxidases, redudases, dehydrogenases, and hydrolases. The enzyme that catalyzes the decomposition of urea is called urease, and so on. 

Hydrolase enzymes catalyze the hydrolysis of a substrate and are most commonly coupled with potentiometericela trodes The pioneering work in this field focussed on de loping an enzyme electrode for the determination of urea. Urease catalyzes the hydrolysis of urea to ammonium and bicarbonate ions according to the reaction detailed below. 

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

Type (820) dinickel complexes offer the opportunity of substrate binding within the bimetallic pocket, and highly preorganized complexes of this type have also been employed as model systems for the urease metalloenzyme (see Section 6.3.4.12.7). The Ni—Ni separation in type (820) complexes can be... 

Enzymes are usually named in terms of the reaction that is catalyzed, commonly adding the suffix -ase to the name of the stoichiometrically converted reactant or substrate. For instance, an enzyme that catalyses the hydrolysis of urea is urease. Enzyme names can only be applied to single enzymes, especially those with termination -ase. For systems that involve the action of two or more enzymes the use of the term should be avoided and the word system should be included. 

An enzyme deposited on the LAPS surface allows one to observe the spatial distribution of a specific substrate. In a urea-selective sensor urease was immobilized on a pH-selective LAPS [75],... 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Both dietary and endogenous ammoniagenic substrates are removed from the intestinal lumen by the osmotic cathartic action of nonabsorbable disaccharides such as lactulose and lactitol. These compounds are currently the main therapeutic agents for chronic HE. The efficacy of oral lactulose for the treatment of HE has been established in controlled trials [41-43]. Besides having a cathartic effect, lactulose lowers the colonic pH as a result of the production of organic acids by bacterial fermentation. The decrease in pH creates an environment that is hostile to the survival of urease-producing intestinal bac-... 

Variation of the nature of the gate electrode results in the different types of FET. For example, in the metal oxide semiconductor FET (MOS-FET) palladium/palladium oxide is used as the gate electrode. This catalyti-cally decomposes gases such as hydrogen sulphide or ammonia with the production of hydrogen ions, which pass into the semiconductor layer. An enzyme may be coated on the palladium, e.g. urease, which catalyses the production of ammonia from urea and thus provides a device for the measurement of this substrate. 

Rates of reaction of urease with deuterated and especially tritiated urea were markedly reduced compared with the rate with the unlabeled ( H) substrate, but usually isotope effects are insignificant biochemically except in rigorous kinetic studies. 

Many enzymes have been named by adding the suffix -ase to the name of their substrate or to a word or phrase describing their activity. Thus urease catalyzes hydrolysis of urea, and DNA polymerase catalyzes the polymerization of nucleotides to form DNA. Other enzymes were named by their discovers for a broad func-... 

Most commonly used enzyme names have the suffix "-ase" attached to the substrate of the reaction (for example, glucosi-dase, urease, sucrase), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]... 

Enzymes are usually impressively specific in their action. The specificity toward substrate is sometimes almost absolute. For many years urea was believed to be the only substrate for the enzyme urease and succinate the only substrate for succinate dehydrogenase. Even after much searching for other substrates, only... 

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... 

The Enzyme Commission catalog (EC 3.5.1.5) lists the urease reaction as urea + 2 H20 = C02 + 2 NH3. Since two C-N bonds are broken it is evident that the stoichiometric relation above is the result of two component reactions. Any conjecture concerning the mechanisms of these reactions and the nature of the intermediates must encompass the action of inhibitors and the spectrum of substrates. Some of the organic inhibitors that have been reported are shown in Table I. The substrates that have been shown to be hydrolyzed are listed in Table II. 

Hydroxyurea is a substrate (95), but the rate at which it is hydrolyzed decreases during the progress of the reaction. The concomitant inhibition disappears as the hydroxyurea is depleted. There is no satisfactory explanation of the events described, and there has been no adequate description of the active site. It has been demonstrated (96) that hydroxyurea added to urease solutions inhibited the hydrolysis of urea and that the extent of inhibition depended both on the order of addition and the time of exposure of the enzyme to the inhibitor. 

Enzymes often are known by common names obtained by adding the suffix -ase to the name of the substrate or to the reaction that they catalyze. Thus, glucose oxidase is an enzyme that catalyzes the oxidation of glucose glucose-6-phosphatase catalyzes the hydrolysis of phosphate from glu-cose-6-phosphate and urease catalyzes the hydrolysis of urea. Common names also are used for some groups of enzymes. For example, an enzyme that transfers a phosphate group from ATP to another molecule is usually called a kinase, instead of the more formal phosphotransferase. ... 

The enzyme urease catalyzes the hydrolysis of urea to form carbamate ion (equation 32). At pH 7.0 and 38 °C, the urease-catalyzed hydrolysis of urea is at least 1014 times as fast as the spontaneous hydrolysis of urea. Jack bean urease is a nickel(II) metalloenzyme502 with each of its six identical subunits containing one active site and two metal ions, and at least one of these nickel ions is implicated in the hydrolysis. It has been suggested503 that all substrates for urease (urea, N-hydroxyurea, 7V-methylurea, semicarbazide formamide and acetamide) are activated towards nucleophilic attack on carbon as a result of O-coordination to the active nickel(II) site as in (155). Nickel(II) ions have been found504 to promote the ethanolysis and hydrolysis of N-(2-pyridylmethyl)urea (Scheme 39) and this system is considered to be a useful model for the enzyme. 

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