Enzyme catalysis reversible reactions

The interest and success of the enzyme-catalyzed reactions in this kind of media is due to several advantages such as (i) solubilization of hydrophobic substrates (ii) ease of recovery of some products (iii) catalysis of reactions that are unfavorable in water (e.g. reversal of hydrolysis reactions in favor of synthesis) (iv) ease of recovery of insoluble biocatalysts (v) increased biocatalyst thermostability (vi) suppression of water-induced side reactions. Furthermore, as already said, enzyme selectivity can be markedly influenced, and even reversed, by the solvent. 

In the classical world (and biochemistry textbooks), transition state theory has been used extensively to model enzyme catalysis. The basic premise of transition state theory is that the reaction converting reactants (e.g. A-H + B) to products (e.g. A + B-H) is treated as a two-step reaction over a static potential energy barrier (Figure 2.1). In Figure 2.1, [A - H B] is the transition state, which can interconvert reversibly with the reactants (A-H-l-B). However, formation of the products (A + B-H) from the transition state is an irreversible step. 

A more realistic but still relatively simple model of enzyme catalysis includes binding of both substrate and product as described by Equation 11.9. This reaction is characterized by five individual rate constants k and k2, and k4 and k5, correspond to the forward and reverse binding steps of the substrate S and product P to the enzyme E, respectively, while k3 expresses the irreversible chemical conversion at the enzyme active site ... 

The cardinal feature of catalysis is that the equilibrium constant of a chemical reaction is unaffected by the presence of a catalyst. This is true as long as the concentration of the catalyst is insignificant relative to the concentra-tion(s) of the least abundant reactant(s) or product(s). Thus for an uncatalyzed reaction (A B) with rate constants k+ and k- (for the forward and reverse rates, respectively), the equilibrium constant K q = k+lk-. In the presence of catalyst, the rate constants are increased, say to xk+ and yk-, and the new equilibrium constant Keq = xk+lyk- = (xly) k+/k-) = x y)K q. Because the rate enhancement in the presence of enzyme will always be the same for forward and reverse reactions, x = y, such that Ksq must still equal K q. 

Water in oil microemulsions with reverse micelles provide an interesting alternative to normal organic solvents in enzyme catalysis with hydrophobic substrates. Reverse micelles are useful microreactors because they can host proteins like enzymes. Catalytic reactions with water insoluble substrates can occur at the large internal water-oil interface inside the microemulsion. The activity and stability of biomolecules can be controlled, mainly by the concentration of water in these media. With the exact knowledge of the phase behaviom" and the corresponding activity of enzymes the application of these media can lead to favomable effects compared to aqueous systems, like hyperactivity or increased stability of the enzymes. 

Microemulsions with different structures, like micelles, reverse micelles or bicontinuous networks, can be used for several inorganic, organic [72] or catalytic reactions which require a large contact area between oil and water. Besides enzyme catalysis, this can be the formation of nanoparticles [54, 73, 74], hydro-formylation reactions [75] or polymerisations [76-78]. 

The surfactant mass fraction in a microemulsion defines the size of the interfacial area between the water and oil. The reaction rate of organic reactions in microemulsions can be dramatically enhanced by increasing the specific interfacial area [95]. Enzyme catalysis in microemulsions is usually not influenced by the size of the interfacial area because only a small fraction of the reverse micelles are hosting a bio-molecule. Most investigations published so far were made with low enzyme concentrations resulting in a low population of enzymes per reverse micelle. 

For a better understanding of the enzyme catalysis in nature, experimental and theoretical studies characterize the free energy profiles and catalytic efficiencies of enzymes under different conditions, which may define the performance of an enzyme in maintaining a constant flux or a constant pool concentration of the product, working under irreversible or reversible conditions etc. (Albery and Knowles, 1976 Stackhouse et al., 1985 Pettersson, 1992 Somogyi, Welch and Damjanovich, 1984). Only a few enzyme reactions have been analyzed in detail and further experimental investigations are necessary to characterize the enzymes, to draw general conclusions, and to deduce how much their evolution approximated the requirements for optimal catalysis . 

Enzymes and micelles resemble each other with respect to both structure (e.g., globular proteins and spherical aggregates) and catalytic activity. Probably the most common form of enzyme catalysis follows the mechanism known in biochemistry as Michaelis-Menton kinetics. In this the rate of the reaction increases with increasing substrate concentration, eventually leveling off. According to this mechanism, enzyme E and substrate A first react reversibly to form a complex EA, which then dissociates to form product P and regenerate the enzyme ... 

If a very low molar concentration of enzyme is present, and a large excess of nonradioactive fructose is added, the enzyme will catalyze no net reaction but will change back and forth repeatedly between the free enzyme and glucosyl enzyme. Each time, in the reverse reaction, it will make use primarily of unlabeled fructose. The net effect will be catalysis of a sucrose-fructose exchange ... 

In an enzyme reaction, initially free enzyme E and free substrate S in their respective ground states initially combine reversibly to an enzyme-substrate (ES) complex. The ES complex passes through a transition state, AGj, on its way to the enzyme-product (EP) complex and then on to the ground state of free enzyme E and free product P. From the formulation of the reaction sequence, a rate law, properly containing only observables in terms of concentrations, can be derived. In enzyme catalysis, the first rate law was written in 1913 by Michaelis and Menten therefore, the corresponding kinetics is named the Michaelis-Menten mechanism. The rate law according to Michaelis-Menten features saturation kinetics with respect to substrate (zero order at high, first order at low substrate concentration) and is first order with respect to enzyme. 

Although free radical reactions are found less often in solution than in the gas phase, they do occur, and are generally handled by steady state methods. There are also organic and inorganic reactions that involve non-radical intermediates in steady state concentrations. These intermediates are often produced by an initial reversible reaction, or a set of reversible reactions. This can be compared with the pre-equilibria discussed in Section 8.4, where the intermediates are in equilibrium concentrations. The steady state treatment is also used extensively in acid-base catalysis and in enzyme kinetics. 

In all of these reactions the species involved in the reversible reactions are progressively used up with time. However, there are reactions such as acid-base catalysis and enzyme catalysis where one of the species in the reversible reaction is the catalyst, and as such is regenerated so that the total catalyst concentration remains constant throughout the whole of the reaction. In such cases it is often essential to use initial rates or rates at various stages during reaction for analysis. 

The lack of reactivity of the semiquinone per se with either thioredoxin or NADPH shows that it cannot be involved in catalysis. The rapid production of semiquinone by irradiation of partially reduced enzyme is a light-activated disproportionation since it is totally dependent upon the presence of some oxidized enzyme. Enzyme fully reduced by dithionite forms no semiquinone, while enzyme partially reduced by dithionite rapidly forms semiquinone upon irradiation. Furthermore, the light-activated disproportionation of enzyme first reduced with NADPH results in the reduction of NADP. Thus, FAD catalyzes the disproportionation in keeping with the known photosensitizing nature of free flavins. This reaction is reversed slowly (half-time ca. 150 min 25°) in the dark. The semiquinone is rapidly reoxidized by oxygen to yield an enzyme with unaltered spectral and catalytic properties (58). Similar reactions have been very briefly reported for lipoamide dehydrogenase the dark reverse reaction is comparatively rapid, being complete in 30 min (16S). 

Although the actual physical mechanism varies for homogeneous, surface, and enzyme catalysis, the processes all involve reaction of the substrate, X, with the catalyst, C, which is usually reversible, and then further reaction of the catalyst-substrate complex, X C, perhaps with another solute, W, to products, P. Mathematically, the catal5hic equations can often be represented in a general way as... 

Metal cofactors in enzymes may be bound reversibly or firmly. Reversible binding occurs in metal-activated enzymes (e.g., many phosphotransferases) firm (or tight) binding occurs in metalloenzymes (e.g., carboxypeptidase A). Metals participate in enzyme catalysis in a number of different ways. An inherent catalytic property of a metal ion may be augmented by the enzyme protein, or metal ions may form complexes with the substrate and the active center of the enzyme and promote catalysis, or metal ions may function in electron transport reactions between substrates and enzymes. 

A molecular assembly in the inner mitochondrial membrane carries out the synthesis of ATP. This enzyme complex was originally called the mitochon-ilrial ATPase or FjFf,ATPase because it was discovered through its catalysis of the reverse reaction, the hydrolysis of ATP. ATP synthase, its preferred name, emphasizes its actual role in the mitochondrion. It is also called (hmplex V. 

As the concentration of the substrate increases, eventually a saturation point is reached beyond this point, the reaction cannot be further accelerated. The plot above is based on the model of enzyme catalysis expressed in the following chemical equation, where the enzyme (E) reacts with the substrate (S) to form some product or products (P). The rates of forward and reverse enzyme-substrate bonding are expressed as ki and k.i, and the rate of product molecule production is expressed as k2. 

Receptor theory is based on the classical Law of Mass Action as developed by Michaelis and Menten (20) for the study of enzyme catalysis. The extrapolation of classical enzyme theory to receptors is, however, an approximation. In an enzyme-substrate (ES) interaction, the substrate S undergoes an enzyme-catalyzed conversion to a product or products. Because of the equilibrium established, product accumulation has the ability to reverse the reaction process. Alternatively, the latter can be used in other cellular pathways and is thus removed from the equilibrium situation or can act as a feedback modulator (21) to alter the ES reaction either positively or negatively (Equation 10.2). 

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