Enzyme denaturation, activation energy

Craig D B, Arriaga E A, Wong J C Y, Lu H and Dovichi N J 1996 Studies on single alkaline phosphatase molecules reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation—the death of an enzyme J. Am. Chem. See. 118 5245-53... 

Thus, a plot of logio Emax versus the reciprocal of the absolute temperature T is linear with the slope equal to -Ea/2.3 R, where Ea is an empirical quantity called the Arrhenius activation energy and R is the gas constant, 1.98 cal/deg-mol. At higher temperatures the maximal velocity generally is much lower than predicted this deviation is the result of denaturation and inactivation of the enzyme at elevated temperatures. 

ProTherm (16) is a large collection of thermodynamic data on protein stability, which has information on 1) protein sequence and stmcture (2) mutation details (wild-type and mutant amino acid hydrophobic to polar, charged to hydrophobic, aliphatic to aromatic, etc.), 3) thermodynamic data obtained from thermal and chemical denaturation experiments (free energy change, transition temperature, enthalpy change, heat capacity change, etc.), 4) experimental methods and conditions (pH, temperature, buffer and ions, measurement and method, etc.), 5) functionality (enzyme activity, binding constants, etc.), and 6) literature. 

Tnterfacial phenomena play a fundamental role in biological systems. It A is important to know if surface energy and anisotropy affect the conformation of biological macromolecules. Well defined physicochemical models might simplify this problem (1- 8) spread monolayers at the air-water interface exemplify this kind of model. For polypeptides which are introduced as simple models of proteins, no surface denatura-tion of the spread macromolecules occurred (9, 10, 11). Protein structures are too complex to yield direct information about eventual changes of conformation, but one can detect the presence or the disappearance of biological activity—e.g., enzymic activity. The enzyme would be denatured if the conformation were modified by the anisotropy of the interface. 

The activation energy of an enzymatic reaction (typical values 20-60 kj/mol) is far below the activation energy of thermal denaturation (values between 200 and 600 kj/ mol) 45. Therefore, by lowering the temperature, the deactivation rate decreases more rapidly than enzyme activity and more product is obtained during the mean lifetime of the enzyme. In practice, the temperature is lowered until enzyme stability is acceptable or other denaturation effects become dominant. Lowering of reaction temperature is of course limited by the solubilities of the substrates and the freezing point of water. 

The enzyme chymotrypsin provides a good example of the strategies and amino acid side chains used by enzymes to lower the amount of activation energy required. Chymotrypsin is a digestive enzyme released into the intestine that catalyzes the hydrolysis of specific peptide bonds in denatured proteins. It is a member of the serine protease superfamily, enzymes that use a serine in the active site to form a covalent intermediate during proteolysis. In the overall hydrolysis reaction, an OH from water is added to the carbonyl carbon of the peptide bond, and an to the N, tha-eby cleaving the bond (Fig. 8.8). The bond that is cleaved is called the scissile bond. 

Some properties of purified endo-(l- -4)- 3-D-xylanase from the ligniperdous fungus Trametes hirsuta have been investigated. The enzyme was stable between pH 4.0 and 8.0 with optimum activity at pH 5.0-5.5. The temperature optimum was 50 C and the enzyme was stable for up to 30 min at 45 C however, it was denatured at higher temperatures. The for 4-0-methyl-D-glucurono-D-xylan was 6.36 X10" equivalents of D-xylose per litre, the activation energy was 28 kj moF The enzyme (mol. wt. 2.2-2.4X 10 by gel chromatography) was activated by Ca " and inhibited by Ag and Hg. On the basis of the effects of 2-hydroxy-5-nitrobenzyl bromide, iV-bromosuccinimide, and A-acetylimidazole it was assumed that L-tryptophan and possibly L-tyrosine residues influence the enzyme catalysts. 

It has been observed that the rate of enzyme reaction rises with temperature up to a certain maximum above which, thermal inactivation of the enzyme takes place. The inactivation of enzymes by heat is due to the denaturation of the enzyme protein. The effect of the instability of the enzyme, free and in the immobilised state, can be studied by exposing the enzyme to thermal treatment for a defined period prior to measuring its activity at a temperature at which it is stable. Chaubey and co-workers obtained 40 °C as the critical temperature of PPy-polyvinyl sulfonate films immobilised with crosslinked lactate dehydrogenase [123]. The activation energies below and above the critical temperature were found to be 93.3 and 22.4 kj/mole, respectively. 

The rates of enzyme-catalyzed reactions also pass through a maximum as the temperature is varied. The increase at lower temperatures is the normal effect, due to there being an activation energy for the enzyme-catalyzed reaction. Enzymes, however, like all proteins, become denatured at higher temperatures and lose their catalytic activity. 

The reaction then takes place in the active site. The products are less strongly bound and fit the active site less well, so they are released from the enzyme, which is then free to react with another substrate molecule. This is the lock and key model of enzyme activity (Figure 16.31). Enzymes, like other catalysts, work by providing an alternative pathway (mechanism) with a lower activation energy. If the enzyme is heated above body temperature, the enzyme changes shape (it is denatured) and loses its activity. 

The frequency factor A (time" ) is a parameter related to the total number of collisions that take place during a chemical reaction, Ea (kJ mol" ) the energy of activation, R (kJ mol" K" ) the universal gas constant, and T (K) the absolute temperature. From Eq. (12.17) we can deduce that for a constant value of A, a higher Ea translates into a lower k. As discussed previously, at a constant A, the higher the value of ko, the more thermostable the enzyme. Thus, the rate constant of denaturation, ko, and the energy of activation of denaturation, Ea, are useful parameters in the kinetic characterization of enzyme stability. 

Figure 12.16. (a) Increases in the rate constant of denaturation (Fp) of an enzyme as a function of increasing temperature, (b) Arrhenius plot used in determination of the energy of activation ( ) of denaturation for the enzyme. 

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