Protein denaturation, activation energy

The process of cooking involves a complicated series of chemical reactions, each of which proceeds with a rate constant of k. When boiling an egg, for example, the rate-limiting process is denaturation of the proteins from which albumen is made. Such denaturation has an activation energy Ea of about 40 kJ mol 1. 

On the other hand, the observation that unfolded proteins undergo irreversible chemical destruction at these extremely high temperatures would mean that a real equilibrium between native and unfolded state cannot exist in these hyperthermophiles. Once unfolded, the proteins become irreversibly denatured. One might speculate that the strategy of protein stabilization at these temperatures aims mainly at an increase of the activation energy of unfolding, i.e., at a retardation of the unfolding process. 

What guides proteins to their native folded state The answer to this question Initially came from In vitro studies on protein refolding. Thermal energy from heat, extremes of pH that alter the charges on amino acid side chains, and chemicals such as urea or guanidine hydrochloride at concentrations of 6-8 M can disrupt the weak noncovalent Interactions that stabilize the native conformation of a protein. The denaturation resulting from such treatment causes a protein to lose both Its native conformation and Its biological activity. 

The kinetics of denaturation of peptide and protein drugs have not been extensively treated. Some studies on the temperature dependence of denaturation rates have been reported. A kinetic study of the denaturation of G actin in solution using DSC yielded linear Arrhenius plots, from which values for the activation energy and the frequency factor of 23 1 kJ/mol (55.2 kcal/mol) and 76.8 sA respectively, were obtained (Fig. 215).889 Linear... 

The size of a molecule is an important feature because proteins form multiple contacts with the surface (e.g., 77 contact points in the case of the albumin molecule and 703 contact points in the case of the fibrinogen molecule adsorbed on silica [10]). Multipoint binding usually causes adsorption irreversibility having a dynamic nature in the absence of irreversible denaturation. The rates of desorption are, as a rule, much lower than those of adsorption, and in many cases it is virtually impossible to attain the equilibrium state desorbing the adsorbed protein [11]. In other words, the formation of one or several bonds with the surface increases the probability of adsorption of neighboring sites of the same molecule. On the other hand, the desorption of a protein molecule requires the simultaneous rupture of a large number of bonds and, for kinetic reasons, equilibrium is not attained [12-14], This corresponds to a considerable difference between the activation energies for the adsorption and desorption processes [15,16],... 

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. 

The denaturation of proteins is a process in which the whole structure becomes less highly ordered. The rate at which it occurs has an extraordinarily high temperature coefficient which corresponds to an enormous activation energy. This fact is important and shows various things. In the first place, the high value of E points to a not inconsiderable stability of the ordered structure itself. Secondly, the fact that the rate of reaction is not thereby reduced to a low value necessitates the conclusion that the non-exponential factor of the denaturation process is very high. In other words, the entropy of activation is great, so that the transition state is much less ordered... 

The validity of this linear extrapolation model (LEM) was proven for monomeric proteins in many cases. Often the LEM is applied even without considering its applicability with the result that the m value has become generally recognized as a standard empirical parameter. The strength of the LEM lies in its simplicity. It is not necessary to calculate denaturant activities, which is an additional source of errors as in the case of the Tanford binding model. Also, the LEM has only one parameter (m), whereas the binding model has two (A and k) if one extrapolates the free energy toward zero denaturant concentration with the function... 

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