Substrate desolvation

Borole, A. P. Cheng, C. L., and Davison, B. H., Substrate Desolvation as a Governing Factor in Enzymatic Transformations of PAHs in Aqueous-Acetonitrile Mixtures. Biotechnology Progress, 2004. 20(4) pp. 1251-1254. 

This article will describe the different chemical strategies used by enzymes to achieve rate acceleration in the reactions that they catalyze. The concept of transition state stabilization applies to all types of catalysts. Because enzyme-catalyzed reactions are contained within an active site of a protein, proximity effects caused by the high effective concentrations of reactive groups are important for enzyme-catalyzed reactions, and, depending on how solvent-exposed the active site is, substrate desolvation may be important also. Examples of acid-base catalysis and covalent (nucleophilic) catalysis will be illustrated as well as examples of "strain" or substrate destabilization, which is a type of catalysis observed rarely in chemical catalysis. Some more advanced topics then will be mentioned briefly the stabilization of reactive intermediates in enzyme active sites and the possible involvement of protein dynamics and hydrogen tunneling in enzyme catalysis. 

Acid/Base Catalysis In Enzymatic Reactions Nucleophilic Catalysis in Enzymatic Reactions Substrate Desolvation... 

Finally, the beam — composed mainly of single substrate and solvent molecules and very small clusters — is passed through a heated wire grid, where the last declustering and desolvation occurs, leaving a beam of substrate molecules. 

The equilibrium binding constant for this 1 1 association is Xu = ki/lLi. The Xu values were measured spectrophotometrically, and the rate constants were determined by the T-jump method (independently of the X,j values), except for substrate No. 6, which could be studied by a conventional mixing technique. Perhaps the most striking feature of these data is the great variability of the rate constants with structure compared with the relative insensitivity of the equilibrium constants. This can be accounted for if the substrate must undergo desolvation before it enters the ligand cavity and then is largely resolvated in the final inclusion complex. ... 

Destabilization of the ES complex can involve structural strain, desolvation, or electrostatic effects. Destabilization by strain or distortion is usually just a consequence of the fact (noted previously) that the enzyme is designed to bind the transition state more strongly than the substrate. When the substrate binds, the imperfect nature of the fit results in distortion or strain in the substrate, the enzyme, or both. This means that the amino acid residues that make up the active site are oriented to coordinate the transition-state structure precisely, but will interact with the substrate or product less effectively. 

Destabilization may also involve desolvation of charged groups on the substrate upon binding in the active site. Charged groups are highly stabilized in... 

FIGURE 16.5 Substrates typically lose waters of hydration in the formation of the ES complex. Desolvation raises the energy of the ES complex, making it more reactive. 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

The active site of enzymes usually are located in clefts and crevices in the protein. This design effectively excludes bulk solvent (water), which would otherwise reduce the catalytic activity of the enzyme. In other words, the substrate molecule is desolvated upon binding, and shielded from bulk solvent in the enzyme active site. Solvation by water is replaced by specific interactions with the protein (Warshel et al., 1989). 

The reaction of l-fluoro-2,4,6-trinitrobenzene and 2,4-dimethoxyaniline, in cyclohexane, shows a negative activation enthalpy274 (—SOkJmoU1), in agreement with a desolvative association mechanism in which the nucleophile competes with the solvent in associating with the substrate in an equilibrium preceding the substitution process. 

A potential factor for enhancing the effectiveness of catalyzed reactions. The relative importance of this factor will depend on the polarity of the substrate (s), transition state, and reaction product(s). The energy associated with desolvation of substrates must be compensated for by the binding interactions between the substrates and the enzyme. 

Following the trend towards lower carbonyl IR stretch frequencies, branching alpha to the amide carbonyl (Table 5, entries 53, 54, 62, 63 and 65) affects the shifts for mutagens and hydroxamic esters similarly and causes a marked downfield shift of up to 6 ppm relative to the acetamide substrate (Table 5, entry 60). These effects, as well as the smaller than expected downfield shift with ferf-butyl and neopentyl side chains are, as with the Ai-chlorohydroxamic esters, due to the combined influence of a stabilizing alkyl inductive effect together with destabilizing desolvation of the polar form of the amide carbonyl ". 

Fig. 1. Construction of a computational model for TauD. (A) the solvated TauD enzyme (PDB code 1GY9, solvating water molecules in red) (B) the desolvated enzyme (C) the active site with the substrate and a-ketoglutarate bound to the iron centre, and the most important amino acids in the first and second coordination sphere (D) a minimal model for TauD including only the first coordination sphere and the substrate.

Second, formation of weak bonds between substrate and enzyme also results in desolvation of the substrate. Enzyme-substrate interactions replace most or all of the hydrogen bonds between the substrate and water. Third, binding energy involving weak interactions formed only in the reaction transition state helps to compensate thermodynamically for any distortion, primarily electron redistribution, that the substrate must undergo to react. 

Strain and stress in enzymes arise from several different causes. We have seen in this chapter, and we shall see further in Chapters 15 and 16, that stress and strain may be divided into two processes, substrate destabilization and transition state stabilization. Substrate destabilization may consist of steric strain, where there are unfavorable interactions between the enzyme and the substrate (e.g., with proline racemase, lysozyme) desolvation of the enzyme (e.g., by displacement of two bound water molecules from the carboxylate of Asp-52 of lysozyme) and desolvation of the substrate (e.g., by displacement of any bound water molecules from a peptide28). Transition state stabilization may consist of the presence of transition state binding modes that are not available for the... 

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