Enzymes urease reaction

Urea is a diagnostic indicator for kidney function. Urea can be potentiometrically determined following the enzymatic reaction of the enzyme urease (reaction [I]) coupled with a variety of potentiometric transducers such as the pC02 electrode, the PNH3 electrode, the pH electrode, and the PNH4 electrode ... 

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. 

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

Urea broadcast into the ricefield floodwater is hydrolysed to ammonium, bicarbonate and hydroxyl ions the reaction is catalysed by the enzyme urease ... 

The formation and excretion of urea is the primary mechanism by which excess nitrogen, in the form of ammonia, is removed from the body. Surprisingly, it was found that the actual rate of urea synthesis exceeded considerably the rate of excretion of the urea. The interesting question, therefore, is what is the fate of this lost urea The answer is that urea enters the large intestine, where it is degraded by microorganisms that possess the enzyme urease, which catalyses the reaction ... 

Rate Enhancement by Urease The enzyme urease enhances the rate of urea hydrolysis at pH 8.0 and 20 °C by a factor of 1014. If a given quantity of urease can completely hydrolyze a given quantity of urea in 5.0 min at 20 °C and pH 8.0, how long would it take for this amount of urea to be hydrolyzed under the same conditions in the absence of urease Assume that both reactions take place in sterile systems so that bacteria cannot attack the urea. 

Phase transition in gels in response to biochemical reactions [27,28]. Polymer gels were synthesized in which an enzyme (urease) or a biologically active protein (lectin) was immobilized. The volume phase transitions were observed in such gels when biochemical reactions took place. Such mechano-biochemical gels will be used in devices such as, sensors, selective absorbers, and biochemically controlled drug release. 

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. 

This reaction is catalyzed by a highly specific enzyme, urease. Urease is present in a number of bacteria and plants. The most common source of the enzyme is jack bean or soybean. Urease was the first enzyme that was crystallized. Sumner, in 1926, proved unequivocally that enzymes are protein molecules. 

Enzyme-catalyzed reactions are used for the determination not only of substrates but also of the enzymes as well as activators and inhibitors of the enzymatic reaction. Substrate concentrations can be measured also by nonkinetic methods by allowing a reaction to proceed to completion before making measurements. Enzymatic methods of analysis have been reviewed by Guilbault. He listed over 150 enzymes with their sources many of these are now available in purified form with high specific activity. Urease was the first to be obtained in pure crystalline form. ... 

In developing some of the elementary principles of the kinetics of enzyme reactions, we shall discuss an enzymatic reaction that has been suggested by Levine and LaCourse as part of a system that would reduce the size of an artificial kidney. The desired result is the production of an artificial kidney that could be worn by the patient and would incorporate a replaceable unit for the elimination of tte nitrogenous waste products such as uric acid and creatinine, In the microencapsulation scheme proposed by Levine and LaCourse, the enzyme urease would be used in tire removal of urea from ti)e bloodstream. Here, the catalytic action of urease would cause urea to decompose into ammonia and carbon dioxide. The mechanism of the reaction is believed to proceed by the following sequence of elementary reactions ... 

Many chemical reactions in living systems are catalyzed by enzymes. An enzyme is a large protein molecule (typically of molar mass 20,000 g moP or more) with a structure capable of carrying out a specific reaction or series of reactions. One or more reactant molecules (called substrates) bind to an enzyme at its active sites. These are regions on the surface of the enzyme where the local structures and chemical properties will selectively bind a specific substrate so particular chemical transformations of it can be carried out (Fig. 18.18). Many enzymes are quite specific in their active sites. The enzyme urease catalyzes the hydrolysis of urea, (NHzlzCO,... 

Life as we know it would be impossible without the astonishing characteristics of enzymic catalysis. This catalysis is not only highly efficient, so that reactions may proceed at low temperature and at neutral pH with the speed required by living cells, but it exhibits also a remarkable specificity. Let us cite two typical examples First, the enzyme urease catalyzes the hydrolysis of urea but of no other compound (1). Second, the catalytic action is frequently restricted to one of the antipodes of optically active substrates. Thus, chymotrypsin will catalyze the hydrolysis of acylated L-tyrosinamides, but will not catalyze the reaction of the corresponding derivatives of D-tyrosine (2). 

The dramatic increases in reaction rates that occur in enzyme-catalyzed reactions can be seen for representative systems in the data given in Table 2.2.4 The hydrolysis of the representative amide benzamide by acid or base yields second-order rate constants that are over six orders of magnitude lower than that measured for ben-zoyl-L-tyrosinamide in the presence of the enzyme a-chymotrypsin. An even more dramatic rate enhancement is observed for the hydrolysis of urea The acid-catalyzed hydrolysis is nearly 13 orders of magnitude slower than hydrolysis with the enzyme urease. The disprotionation of hydrogen peroxide into water and molecular oxygen is enhanced by a factor of 1 million in the presence of catalase. 

The first applications of enzymes in bioanalytical chemistry can be dated back to the middle of nineteenth century, and they were also used for design of first biosensors. These enzymes, which have proved particularly useful in development of biosensors, are able to stabilize the transition state between substrate and its products at the active sites. Enzymes are classified regarding their functions, and the classes of enzymes are relevant to different types of biosensors. The increase in reaction rate that occurs in enzyme-catalyzed reactions may range from several up to e.g. 13 orders of magnitude observed for hydrolysis of urea in the presence of urease. Kinetic properties of enzymes are most commonly expressed by Michaelis constant Ku that corresponds to concentration of substrate required to achieve half of the maximum rate of enzyme-catalyzed reaction. When enzyme is saturated, the reaction rate depends only on the turnover number, i.e., number of substrate molecules reacting per second. 

The enzyme urease, which catalyzes the hydrolysis of urea, is widely used to determine urea in blood. Details of this application are given in Feature 29-3 on page 901. The Michaelis constant for urease at room temperature is 2.0 mM, and k2 = 2.5 X 10 s at pH 7.5. (a) Calculate the initial rate of the reaction when the urea concentration is 0.030 niM and the urease concentration is 5.0 p,M, and (b) find v, ax-... 

The determination of urea in blood and urine is frequently carried out by measuring the rate of hydrolysis of urea CO(NH2)2 in the presence of the enzyme urease. The equation for the reaction is... 

The sensitive part of an electrode is covered with a membrane on which the enzyme is immobilized in immunocomplexes. The enzyme-catalyzed reaction takes place near the sensor (Mattiasson and Nilsson, 1977). The method is as fast as the thermometric assay but less sensitive. Electrode-based EIA using urease conjugates have been reviewed by Meyerhoff and Rechnitz (1980). This method has reasonably low detection limits. These promising potentiometric EIA are discussed by Boiteux et al. (1981) and Gabauer and Rechnitz (1982). 

The best-known enzyme electrode is that used to analyze for urea in blood. The enzyme urease is immobilized in a polyacrylamide hydrophilic gel and fixed at the bottom of a glass electrode whose characteristics make it an NH4 ISE. Alternatively, the ISE can be a composite system designed to detect NH3. In the presence of the enzyme, urea is hydrolyzed according to the reaction... 

While the majority of enzyme electrodes fabricated have been rather large devices, there have been some recent reports concerning the development of miniaturized and even microsensors. For example, MeyerhoflF (M5) prepared an essentially disposable urea sensor (tip diameter 3 mm) by immobilizing urease at the surface of a new type of polymer-membrane electrode-based ammonia sensor (see Fig. 4). Alexander and Joseph (Al) have also prepared a new miniature urea sensor by immobilizing urease at the surface of pH-sensitive antimony wire. Similarly, lannello and Ycynych (II) immobilized urease on a pH-sensitive iridium dioxide electrode. In these latter investigations, ammonia liberated from the enzyme-catalyzed reaction alters the pH in the thin film of enzyme adjacent to the pH-sensitive wire. 

Besides these conventional reactors with spherical immobilizates, urease has also been immobilized inside nylon tubing and pipette tips ( enzyme pipette , Sundaram and Jayonne, 1979), on nylon fibers ( enzyme brush , Raghavan et al., 1986), and on the surface of a magnetic stirrer (Guilbault and Starklov, 1975). The urease reaction was in each case carried out at optimal pH after removal of the immobilized enzyme NH3 was assayed electrochemically or photometrically according to Berthelot s method. 

In water at room temperature, the rate constant for the uncatalyzed reaction is 3X10 ° s . Under the same conditions in the presence of the enzyme urease... 

Table 10.1 gives values of rate constants, activation energies, and frequency factors for three enzyme-catalyzed reactions. For comparison, the values for other catalysts are included. Note that molecule for molecule, the enzymes are much more effective catalysts than the nonbiological catalysts. In urease and catalase this higher effectiveness is related to a much smaller activation energy, which is true for a number of other enzyme systems. Enzymes evidently exert their action by allowing the process to occur by a much more favorable reaction path. 

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