Urease substrate specificity

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. 

For this reason, the sudden change in color of the indicator phenolphthalein can serve to verify the hydrolysis (Experiment 19.4). Structurally related substances like thiourea, methylurea, or semicarbazide are, by contrast, not decomposed. This is an indication of the high substrate specificity of urease. 

The quotient k JK represents the apparent rate coefficient of a second-order reaction whose rate density is determined by the frequency of (effective) collisions between enzyme and substrate molecules. For the same concentrations of enzyme and substrate, respectively, the catalytic efficiency can be described by the quotient koJKM- With the help of the values of k /K, we can investigate which among several substrates the enzyme prefers is therefore a measure of the substrate specificity of an enzyme where high values indicate high specificity. Typical values lie between 10 and 10 mol L s . Table 19.1 is a compilation of characteristic quantities of the enzymes urease and catalase. 

Apart from being specific for a particular reaction, enzjmies show varying degrees of substrate specificity. This ranges from the absolute specificity shown by urease, which has urea as its one and only substrate, to group specificity in which an enzyme will act upon a general type of substrate, e.g. alcohols, esters or peptide bonds. The lipases have a broad specificity and will act on the esters of most fatty acids, while hexokinase catalyses the phosphorylation of a variety of aldohexoses. 

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

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],... 

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... 

This reaction differs from the spontaneous hydrolysis of urea to ammonium and cyanate, which is 1024 times slower. Urease is a highly specific and efficient enzyme. Its other substrates, of which the best are semicarbazide, formamide, acetamide, and A-methylurea, are hydrolyzed at very low rates [39],... 

The chemical nature of enzyme was controversial for a long time, until Buchner succeeded in isolating an enzyme system (zymase) from yeast in a cell-free extract in 1897.2) Urease was then crystallized by Sumner in 1926,3) followed by crystallization of several proteolytic enzymes by Northlop and his colleagues. At present the chemical nature of enzyme is defined as a protein with catalytic activity based on the specific activaiton of its substrate. However, this definition has been somewhat open to debate since a catalytic RNA, ribozyme, was discoved in 1982. 

Enzymes are substances that react very selectively with a substrate in a very specific reaction. Their immobilization on a membrane which is then placed over an electrode in a solution together with the substrate to be determined leads to reaction products that can be detected at the electrode covered by the membrane. An example is the degradation of urea by urease with an internal sensor element (i.e. ion-selective electrode) sensitive to ammonium ion ... 

Such interference falls into two classes competitive substrates and substances that either aaivate or inhibit the enzyme. With some enzymes, such as urease, the only substrate that reacts at reasonable rate is urease hence, the urease-coated electrode is specific for use (59, 165). Likewise, uricase acts almost specifically on uric acid (167), and aspartase on aspartic acid (8, 168). Others, such as penicillinase and amino oxidase, are less specific (63,169,170). Alcohol oxidase responds to methanol, ethanol, and allyl alcohol (171, 172). Hence, in using electrodes of these enzymes, the analyte must be separated if two or more are present (172). Assaying L-amino acids by using either the decarboxylative or the deaminating enzymes, each of which acts specifically on a different amino... 

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. ... 

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). 

There are over 300 enzymes available commercially, and many of these can be used in one way or another for analytical purposes. One of methods of use involves the determination of an analyte or substrate by means of the enzyme which reacts specifically with that substrate. Examples are 1. Glucose and glucose oxidase, O2 released, measurement by O2-ISE 2. Urea and urease, NHs and CO2 released, measurement by an NH4 - or CO2-ISE 3. Pectin and pectin-esterase, titration of H+ released. In chnical work, the opposing approach is often used, and the amount of an enzyme determined by adding the proper substrate to a solution of the enzyme and measuring with an ion-selective electrode a product of the reaction. Early work in this area, and in that of immobihsed enzyme electrodes, was carried out by Katz and Rechnitz [15,16], Guilbault and his co-workers [17] and Guilhault and Montalvo [18]. 

Absolute specificity Absolute specificity refers to an enzyme that catalyzes only one reaction on only one substrate, such as urease ... 

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