Urease, rate constant

A good example of the range of parameters available from flow calorimetric data can be found from the study of enzyme/substrate systems. The kinetic nature of enzyme systems has been previously described by Michaelis and Menten. In the treatment discussed here, the parameters sought are the enthalpy, rate constant, Michaelis constant and the enzyme activity. The following example describes a study on the well-known enzyme substrate system, urea/urease. 

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. 

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. 

The enzyme urease catalyzes the reaction of urea, (NH2CONH2), with water to produce carbon dioxide and ammonia. In water, without the enzyme, the reaction proceeds with a first-order rate constant of 4.15 X 10 s at 100 °C. In the presence of the enzyme in water, the reaction proceeds with a rate constant of 3.4 X 10 s at 21 °C. (a) Write out the balanced equation for the reaction catalyzed by urease, (b) Assuming the collision fector is the same for both situations, estimate the difference in activation energies for the uncat-... 

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 (pronounced yMr-ee-ase ), the rate constant increases lO -fold, to 3x10 s Enzymes are also extremely specific urease catalyzes only this hydrolysis reaction, and no other enzyme does so. 

A 50 ml aliquot of the urea solution is pre-thermostated to the operational temperature of the calorimeter for these experiments the calorimeter is housed in a constant temperature environment and operated at 25 °C. The urea solution is then run in a continuous loop, at a known flow rate, until a stable baseline is achieved. This solution is then inoculated with 4.55 ml of a standard, fixed concentration, urease solution (also buffered to pH 7.0 and pre-thermostated) and the resulting calorimetric output recorded as a function of time. This is repeated for all concentrations of urea. Figure 5 shows a selection of typical calorimetric outputs for this enzyme system. 

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 interior pellet pH is a function of the urease loading and the urea concentration profile in the pellet. The for urease hydrolysis of urea is 2.9 mM, [29] so with 0.01 M (10 mM) urea, we are initially consuming urea at approximately 78% of Fmax at the surface of the pellet. Increasing the bulk concentration of urea will result in increased ammonia production and an increase in the interior pellet pH. Depending upon whether the interior pH is above or below the pH for optimum XI activity, an increase in interior pH will decrease or increase the rate of xylose isomerization. To achieve optimal isomerization in the co-immobilized pellet system, the urea concentration in the bulk solution can be optimized for a specific urease loading and should be maintained at a constant concentration throughout the isomerization to allow maximal, constant XI activity. 

In order to determine the catalytic characteristics of the encapsulated enzymes, we obtained the dependences of the stationary rate of substrate conversion on the substrate concentration. As an example, the curves of saturation of urease with urea in the reaction of urea decomposition are depicted in Figure 5. It can be seen that the dependences for urease in microcapsules, are generally similar to those for free enzyme, except small differences in the affinity constants. In particular, the Michaelis constant AM with respect to urea is 7.1 D2.2 mM for urease in microcapsules of eleven and seven layers, whereas the AM for free urease is 2.5 DO. mM. The maximal rate Umax for urease in microcapsules of eleven layers is 20% lower than that for urease in microcapsules of seven layers. The AM with respect to pyruvate for microcapsules containing LDH was not different from for free enzyme. 

A similar activation of urea hydrolysis is seen when pH measurements are made in the cytosensor and the rate of alkalinization correlated with urease activity, as shown in the figure on the opposite page. This also differs from previous investigations, because acid is constantly added to the organisms, and inactivation of the mechanism for enhancement of urease activity does not occur. There is approximately a 40-fold activation of urease activity as the pH falls to below 4.0. At higher pH, in the small volume of the chamber, urease activity is able to elevate chamber pH very rapidly hence, urease activity is only transient. At a pH of 3.0, the level of acid is sufficient to require significant levels of urease activity to attempt to elevate pH. These... 

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