Catalytic enzymes temperature influence

Two different types of enzymatic time-temperature integrators are described. The first, under the tradename of I-point, is based on a lipase-catalyzed hydrolysis reaction (125). The lipase is stored in a nonaqueous environment containing glycerol. The indicator contains two components that are mixed when the indicator is activated. The operating principle is as follows Upon activation, two volumes of reagents are mixed with each other. Lipase is thereby exposed to its substrate, here a triglyceride. At low temperatures there will be almost no hydrolytic reaction. As the temperature increases, hydrolysis accelerates and protons are liberated. A pH indicator is dissolved in the system. The indicator is selected to shift color after a certain amount of acid has been liberated by the enzyme-catalyzed process. Since the catalytic activity is influenced both by temperature and time, this indicator strip is said to be a time-temperature integrator. 

The catalytic activity of enzymes is influenced by numerous factors. The most important are substrate concentration, enzyme concentration, temperature, and pH. 

In the measurement of enzyme activity, a high substrate concentration that is greatly in excess of the Km value is always used, and the enzyme sample to be investigated is correspondingly diluted vmder the conditions, the rate of the enzyme-catalyzed reaction depends only on the enzyme concentration, i.e., it is a zero order reaction. Even under conditions of substrate saturation, the measured catalytic activities are influenced by slight differences in reaction conditions, such as the temperature, composition and concentration of the buffer, pH value, nature of the substrate and its concentration, coenzymes, and protein content in the sample. Therefore, the results of measurement of the catalytic activity of an enzyme are in principle method dependent direct comparison of the results between laboratories is made difficult by the use of different methods in different laboratories. 

Enzyme Sta.bihty, Loss of enzyme-catalytic activity may be caused by physical denaturation, eg, high temperature, drying/freezing, etc or by chemical denaturation, eg, acidic or alkaline hydrolysis, proteolysis, oxidation, denaturants such as surfactants or solvents, etc. pH has a strong influence on enzyme stabiHty, and must be adjusted to a range suitable for the particular enzyme. If the enzyme is not sufficiendy stable in aqueous solution, it can be stabilized by certain additives a comprehensive treatment with additional examples is available (27). 

The catalytic properties of enzymes, and consequently their activity (see p. 90), are influenced by numerous factors, which all have to be optimized and controlled if activity measurements are to be carried out in a useful and reproducible fashion. These factors include physical quantities (temperature, pressure), the chemical properties of the solution (pH value, ionic strength), and the concentrations of the relevant substrates, cofactors, and inhibitors. 

The ratio of the turnover number (i.e., Emax/[Etotai]) to the Xn, value of a substrate in a particular enzyme-catalyzed reaction. When kcat and are the true steady-state parameters, this ratio (or the ratio Emax/T m) is an excellent gauge of the specificity of the enzyme for that substrate. The larger the ratio, the more effective that substrate is used by the enzyme under study. In addition, the effects of a number of mechanistic probes of enzyme action on this ratio (for example, pH effects, isotope effects, temperature effects, the influence of various modifiers, etc.) can provide much information on the catalytic and binding mechanism. See... 

In the case of most enzymic transformations the reaction rate can be described as a hyperbolic function of the concentration of substrate the characteristic parameters of these hyperboles are the and the KM values, which can be determined easily by different linearized plots. Different factors such as temperature, pH, chemical modification of the functional groups in the side chains of the protein, reversible inhibitors, activators, allosteric effectors, influence the catalytic activity of the enzymes. 

Molded Dry Chemistry. In general, most enzymes are very fragile and sensitive to pH. solvent, and elevated lemperaiurts. The catalytic activity of most enzymes i> reduced dramatically ils the temperature is increased, Typi cal properties of diagnostic enzymes are given in Table 1. t he presence of ionic salts and other chemicals can considerably influence enzyme stability. To keep or sustain enzymatic activity, the redox centers must remain intact. The bulk of the enzyme, polymeric in composition, is an insulaior. thus. altering ii does not reduce the enzyme s catalytic activity, li... 

Xanthine is converted to uric acid at the molybdenum center of the enzyme, and the electrons are removed from the enzyme by oxidation of the flavin center. From early reductive titrations of xanthine oxidase with sodium dithionite, it was proposed that reducing equivalents were equilibrated among the four redox-active centers (Mo-co, two separate Fe2S2 centers, flavin) at a rate that was rapid relative to the overall catalytic rate of substrate turnover (243). Under such conditions, the flux of reducing equivalents through the enzyme should be influenced by the relative reduction potentials of the redox centers involved (244). Any effects of pH and temperature on the reduction potentials of individual redox components would affect the apparent rates of intramolecular transfer of the enzyme. 

Another factor that greatly influences the rate of enzyme-catalyzed reactions in addition to pH and temperature is the presence of an inhibitor. As follows, we need to mention the effect that different inhibitors have on the rate. The three most common types of reversible inhibition occuring in enzymatic reactions are competitive, uncompetitive, and noncompetitive [3]. The enzyme molecule is analogous to the heterogeneous catalytic surface in that it contains active sites. When competitive inhibition occurs, the substrate and inhibitor are usually similar molecules that compete for the same site on the enzyme. The resulting inhibitor-enzyme complex is inactive. The reactions can be developed as follows (Eq. 4-1 and Eqs. 4-12 to 4-3). 

The temperature dependencies of catalytic activity are qualitatively similar for all enzymes (Fig. 4.1). For any enzyme, there exists a so-called point of temperature optimum, i.e., the temperature of maximal catalytic activity. At the low-temperature branch of the temperature dependence, the increase in the enzyme activity with rising temperature is usually explained within the Arrhenius mechanism. This viewpoint, however, seems to be oversimplified. The temperature rise not only enhances the Boltzmann factor, increasing the probability of overcoming the potential barrier, but also influences the structural properties of an enzyme molecule. According to the majority of textbooks on biochemistry, the decrease in catalytic activity at high temperatures is due to the reversible inactivation of an enzyme or its denaturation occurring with a sufficient increase in the temperature. If the latter factor dominates, the value of the temperature optimum will be determined in prac-... 

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