Enzymes properties

Each enzyme has a working name, a specific name in relation to the enzyme action and a code of four numbers the first indicates the type of catalysed reaction the second and third, the sub- and sub-subclass of reaction and the fourth indentifies the enzyme [18]. In all relevant studies, it is necessary to state the source of the enzyme, the physical state of drying (lyophilized or air-dried), the purity and the catalytic activity. The main parameter, from an analytical viewpoint is the catalytic activity which is expressed in the enzyme Unit (U) or in katal. One U corresponds to the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute whereas one katal (SI unit) is the amount of enzyme that converts 1 mole of substrate per second. The activity of the enzyme toward a specific reaction is evaluated by the rate of the catalytic reaction using the Michaelis-Menten equation V0 = Vmax[S]/([S] + kM) where V0 is the initial rate of the reaction, defined as the activity Vmax is the maximum rate, [S] the concentration of substrate and KM the Michaelis constant which give the relative enzyme-substrate affinity. 

The specific properties of a series of enzymes of interest for various applications and intensively investigated for immobilization are listed in Table 15.1. 

Glucose oxidase (GOD) Aspergillus niger VII redox glucose 

Peroxidase (HRP) horseradish redox/H202 amine, phenol, organics oxidation 

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Chemically, the membrane is known to consist of phospholipids and proteins, many of which have enzymic properties. The phospholipid molecules are arranged in a bimolecular layer with the polar groups directed outwards on both sides. The structures of some phospholipids found in bacteria are shown in Fig. 1.6. Earlier views held that the protein part ofthe membrane was spread as a continuous sheet on either side ofthe... 

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

Progress in molecular biology has provided a new perspective. Techniques such as the polymerase chain reaction and single-strand conformation polymorphism analysis have greatly facilitated the molecular analysis of erythroenzymopathies. These studies have clarified the correlation between the functional and structural abnormalities of the variant enzymes. In general, the mutations that induce an alteration of substrate binding site and/or enzyme instability might result in markedly altered enzyme properties and severe clinical symptoms. 

Morley, K.L. and Kazlauskas, R.J. (2005) Improving enzyme properties when are closer mutations better Trends in Biotechnology, 23, 231-237. 

L. Hernandez, J. Arrieta, C. Menendez, R. Vazquez, A. Coego, V. Suarez, G. Selman, M. F. Petit-Glatron, and R. Chambert, Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane, Biochem. J., 309(Pt 1), (1995) 113-118. 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

Greater purification could be achieved, usually on a smaller scale, if the desired protein had easily measurable properties (e.g. was an enzyme), so that the specific activity of the product and extent of purification could be estimated. Well into the 1950s any enzyme required in a laboratory had first to be isolated by those needing it. With increased knowledge of enzyme properties two further methods of purification were commonly tried heat denaturation of contaminating proteins (with the incidental discovery of some remarkably heat-resistant enzymes), and protein precipitation at the iso-electric point. 

Numerous different immobilization methods have been reported that take advantage of various enzyme properties such as size, chemically reactive functionality, ionic groups or hydrophobic domains.Based on these properties, enzyme immobilization can be split into three main classes (which are also applicable to the immobilization of cell cultures) ... 

Actin, the most abundant protein in eukaryotic cells, is the protein component of the microfilaments (actin filaments). Actin occurs in two forms—a monomolecular form (C actin, globular actin) and a polymer (F actin, filamentous actin). G actin is an asymmetrical molecule with a mass of 42 kDa, consisting of two domains. As the ionic strength increases, G actin aggregates reversibly to form F actin, a helical homopolymer. G actin carries a firmly bound ATP molecule that is slowly hydrolyzed in F actin to form ADR Actin therefore also has enzyme properties (ATPase activity). 

The enzymatic approach to the synthesis of carbohydrates and their precursors is practical for certain types of sugars. The next stage of our investigation will be to focus on the modification of these readily available sugars to agents of interest. Improvement of enzyme properties for synthetic application is also of interest. 

Enzyme Properties. The two isolated veratryl alcohol oxidases had very similar properties (Table I). The difference in isoelectric points might be accounted for by aspartate content all other amino acid contents except glycine were the same within experimental error (5%). The specific activities (veratryl alcohol as substrate) were significantly different, but both enzymes contained a flavin prosthetic group (25) and converted one molecule of oxygen to one molecule of hydrogen peroxide during alcohol oxidation. 

The enzyme properties reported above are similar to those of an aromatic alcohol oxidase from Polystictus versicolor (27). However, the latter enzyme had a different substrate specificity and the cultures did not produce laccase. 

Kaltenbach, M. et al., Flavonoid hydroxylase from Catharanthus roseus. cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants. Plant J., 19, 183, 1999. 

In RMs, enzyme properties will depend on essentially three factors the structure of RMs, the dynamics of RMs, and the distribution of enzyme in RMs. Based on an understanding of these aspects, Bru et al. [183] have developed a model, as summarized below, which simulates the dependence of enzyme activity on water content. 

Resell, C., Femandez-Lafuente, R. and Gnisan, J.M. (1995) Modification of enzyme properties by the use of inhibitors dnring their stabilisation by multipoint covalent attachment. Biocatalysis and Biotransformation, 12, 67-76. 

In Figure 10.1 the time course of thermodynamically and kinetically controlled processes catalysed by biocatalysts are compared. The product yield at the maximum or end point is influenced by pH, temperature, ionic strength, and the solubility of the product. In the kinetically controlled process (but not in the thermodynamically controlled process) the maximum yield also depends on the properties of the enzyme (see next sections). In both processes the enzyme properties determine the time required to reach the desired end point. The conditions under which maximum product yields are obtained do not generally coincide with the conditions where the enzyme has its optimal kinetic properties or stability. The primary objective is to obtain maximum yields. For this aim it is not sufficient to know the kinetic properties of the enzyme as functions of various parameters. It is also necessary to know how the thermodynamically or the kinetically controlled maximum is influenced by pH, temperature and ionic strength, and how this may be influenced by the immobilization of the biocatalysts on different supports. 

Bhagwat AS (1992) Restriction enzymes Properties and use. Methods Enzymol 216 199-224... 

The functional properties are divided according to a completely different pattern. Enzymic properties are functional ones because the action of the catalyst is, per se, a nonnutri-tional one. In certain cases, when enzymes are added to foods as in vivo digestion aids, they might be considered metabolic enhancers. Otherwise, when used externally to prepare products possessing more utile chemical and physical characteristics, the digestive nature is indubitably a functional property. It must be noted, however, that enzyme functionality usually is not studied in conjunction with the other functionalities, but is a distinct and separate branch of biochemistry involved in functional evaluation. Theoretically, a more scientific division of the functional properties could be made into molecular and non-molecular ones. (Enzymic properties would then be a division of the former.) However, traditional lines already have been set and the proposed division is closer to present research d i sci piines. 

Functional Evaluation - An evaluation of the basic physico-chemical properties that influence functionality. (By this definition the functional tests of enzymic properties are functional evaluations.)... 

Nakagawa, T. Nagayama, E Enzymic properties of fish muscle creatine kinase. Comp. Biochem. Physiol. B, 98, 349-354 (1991)... 

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