Enzyme temperature effects

The concept of equilibrium is also important in biochemical processes such as O2/CO2 exchange in hemoglobin (for example, see exercise 5.8 at the end of this chapter), the binding of small molecules to DNA strands (as might occur in the transcription process), and the interaction of substrates and enzymes. Temperature effects are important in protein denaturation process. Clearly, the ideas established in this chapter are widely applicable to all chemical reactions, even very complex ones. 

Calcined [MgAl] LDH was also used to adsorb penicillin G acylase [121]. The calcined LDH phases have porous structures, large specific surface areas and abundant basic sites to bind the enzymes. The effect of varying the composition of the LDH precursor and calcination temperature on the activity of the immobilized enzyme has been reported. In this case, the percentage of immobilized proteins increases up to 88 %. 

A reaction which follows power-law kinetics generally leads to a single, unique steady state, provided that there are no temperature effects upon the system. However, for certain reactions, such as gas-phase reactions involving competition for surface active sites on a catalyst, or for some enzyme reactions, the design equations may indicate several potential steady-state operating conditions. A reaction for which the rate law includes concentrations in both the numerator and denominator may lead to multiple steady states. The following example (Lynch, 1986) illustrates the multiple steady states... 

Cano, M.P., Hernandez, A., and De Ancos, B. 1997. High pressure and temperature effects on enzyme inactivation in strawberry and orange products. J. Food Sci. 62, 85-88. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 340-352 measurement, 63, 365 cryosolvent [catalytic effect, 63, 344-346 choice, 63, 341-343 dielectric constant, 63, 354 electrolyte solubility, 63, 355, 356 enzyme stability, 63, 344 pH measurements, 63, 357, 358 preparation, 63, 358-361 viscosity effects, 63, 358] intermediate detection, 63, 349, 350 mixing techniques, 63, 361, 362 rapid reaction techniques, 63, 367-369 temperature control, 63, 363-367 temperature effect on catalysis, 63, 348, 349 temperature effect on enzyme structure, 63, 348. 

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... 

The study of temperature effects on the reactivation of an enzyme that has been completely unfolded allows one to distinguish between reactivation (referring to kinetic analysis exclusively) and renaturation, the latter of which would reflect both the refolding transition and the formation of misfolded or aggregated byproducts. 

Dixon and Webb provided a useful list of various causes for the shape of the temperature effect seen in enzyme-catalyzed reactions (1) effect on enzyme stability (2) effect on the actual velocity of the reaction (especially on kcat) (3) effect on affinity(ies) of the substrate(s)... 

This dependence on light levels and temperature is believed to be due to the mechanism of production of isoprene in the plant, which involves the enzyme isoprene synthetase and dimethylallyl diphosphate (DMAPP) as a precursor to isoprene (e.g., see Silver and Fall, 1995 and Monson et al., 1995). Either the enzyme, the formation of DMAPP, or both may be light sensitive (Wildermuth and Fall, 1996). The temperature effect has been attributed to effects on the enzyme, increasing its activity initially and then leading to irreversible denaturation (and/or possibly membrane damage) (Fall and Wildermuth, 1998). 

The thermodynamics of these reaction systems have been investigated, resulting in methods to predict the direction of a typical reaction a priori. Furthermore, studies on kinetics, enzyme concentration, pH/temperature effects, mixing, and solvent selection have opened up new perspectives for the understanding, modeling, optimization, and possible large-scale application of such a strategy. 

The temperature effect is much more significant than the pressure effect. For the enzyme stability, a temperature increase above certain levels, depending on the enzyme, results in deactivation of the enzyme. In Table 9.2-2, the residual activities of various enzymes after one hour incubation time, in supercritical CO2 at 150 bar, are given. It is obvious that temperatures over ca. 75°C reduce enzyme activity dramatically. However, no correlation for the stability with the temperature for different types of enzymes is yet available [8-10],... 

A classic text, directed at the food sciences, covering the fundamental principles of enzymology. Chapters covering enzyme purification, pH effects, temperature effects, enzyme inhibitors, and the proteolytic enzymes are particularly relevant to this unit. 

The rate of urea denaturation was inhibited by a variety of anions known to bind to the enzyme in decreasing order of effectiveness, pyrophosphate, 2 -CMP, phosphate, citrate, tartrate, and sulfate (353). This inhibition was greater at pH 5.6 than 7.3. The binding constants were the same as those estimated by inhibition of the enzymic reaction. As with the pH and temperature effects, the anions had no demonstrable effect on the rate of renaturation. 

Enzyme Temperature (°C) Pressure (MPa) Time (h) Effect Reference... 

Almeida, M. C. Ruivo, R. Maia, C. Freire, L. de Sampaio, T. C. Barreiros, S. Novozym 435 Activity in Compressed Gases. Water Activity and Temperature Effects. Enzyme Microb. Technol. 1998, 22, 494 499. 

The effect of temperature satisfies the Arrhenius relationship where the applicable range is relatively small because of low and high temperature effects. The effect of extreme pH values is related to the nature of enzymatic proteins as polyvalent acids and bases, with acid and basic groups (hydrophilic) concentrated on the outside of the protein. Finally, mechanical forces such as surface tension and shear can affect enzyme activity by disturbing the shape of the enzyme molecules. Since the shape of the active site of the enzyme is constructed to correspond to the shape of the substrate, small alteration in the structure can severely affect enzyme activity. Reactor s stirrer speed, flowrate, and foaming must be controlled to maintain the productivity of the enzyme. Consequently, during experimental investigations of the kinetics enzyme catalyzed reactions, temperature, shear, and pH are carefully controlled the last by use of buffered solutions. 

The explanation of Ql(l effects just presented is rather typical of treatments found in most textbooks, in which a relatively simplified thermodynamic explanation, based on energy distribution patterns, is developed to account for effects of temperature on reaction rates. Such treatments of temperature effects, while correct overall, are abstract and nonmecha-nistic—a necessary property of thermodynamic explanations—and will be seen to be incomplete in important ways. In particular, thermodynamic treatments that eschew discussions of underlying mechanisms are unable to provide an explicit account of what steps in an enzyme-catalyzed reaction are rate limiting and, thus, responsible for Qio effects. 

Clark, K.J., F.W. Chaplin, and S.W. Harcum. 2004. Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA. Biotechnol Prog 20 1888-1892. 

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