Enzymes and enzyme-mediated reactions

This section deals with the nature of enzymes and their importance in metabolic control is discussed more fully in Chapter 3. Enzymes are biocatalysts whose key characteristics are as follows  

The majority of biochemical reactions are reversible under physiological conditions of substrate concentration. In metabolism, we are therefore dealing with chemical equilibria (plural). The word equilibrium (singular) signifies a balance, which in chemical terms implies that the rate of a forward reaction is balanced (i.e. the same as) the rate of the corresponding reverse reaction. 

Many chemical reactions (especially those occurring within cells) are theoretically reversible under reasonable conditions of pressure (when gasses are involved, which is rare), temperature and concentration. 

In a closed system, that is one in which there is no addition of V nor any removal of p , the reaction will come to a perfect balance the point of equilibrium . A common misunderstanding of the concept of this point of equilibrium is that it implies an equal concentration of r and p. This is not true. The point of equilibrium defines the relative concentrations of r and p when the rate of formation of p is exactly equal to the rate of formation of r. The point of equilibrium value for a chemical reaction can be determined experimentally. If the starting concentration of the reactant is known, then it follows that the relative concentrations of r and p when equilibrium has been reached must reflect the relative rates of the forward and reverse reactions. For a given reaction, under defined conditions, the point of equilibrium is a constant and given the symbol Keq. 

When the equilibrium concentration of p is greater than the equilibrium concentration of r, we can say that the forward reaction is favoured (faster) and Keq 1  

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By protodetritiation of the thiazolium salt (152) and of 2 tritiothiamine (153) Kemp and O Brien (432) measured a kinetic isotope effect, of 2.7 for (152). They evaluated the rate of protonation of the corresponding yiides and found that the enzyme-mediated reaction of thiamine with pyruvate is at least 10 times faster than the maximum rate possible with 152. The scale of this rate ratio establishes the presence within the enzyme of a higher concentration of thiamine ylide than can be realized in water. Thus a major role of the enzyme might be to change the relative thermodynamic stabilities of thiamine and its ylide (432). 

The activity of many enzymes is pH-dependent because the enzyme may ionize in solution and the biological activity of unionized and ionized forms may be different. In this case, the rate of an enzyme-mediated reaction can be expected to depend on the acidity of the solution. If the enzyme can lose more than one proton as the pH increases (Figure 8.12), the rate of reaction as a function of pH may display a maximum if the forms of the enzyme in strongly acidic or strongly basic solution are inactive, but the intermediate, monoanion, is active. An example of this behavior is provided by fumarase (Figure 8.13). 

In the case of fermentation, the carbon and energy source is broken down by a series of enzyme-mediated reactions that do not involve an electron transport chain. In aerobic respiration, the carbon and energy source is broken down by a series of enzyme-mediated reactions in which oxygen serves as an external electron acceptor. In anaerobic respiration, the carbon and energy source is broken down by a series of enzyme-mediated reactions in which sulfates, nitrates, and carbon dioxide serve... 

Equations 2.26 and 2.27 carmot be solved analytically except for a series of limiting cases considered by Bartlett and Pratt [147,192]. Since fine control of film thickness and organization can be achieved with LbL self-assembled enzyme polyelectrolyte multilayers, these different cases of the kinetic case-diagram for amperometric enzyme electrodes could be tested [147]. For the enzyme multilayer with entrapped mediator in the mediator-limited kinetics (enzyme-mediator reaction rate-determining step), two kinetic cases deserve consideration in this system in both cases I and II, there is no substrate dependence since the kinetics are mediator limited and the current is potential dependent, since the mediator concentration is potential dependent. Since diffusion is fast as compared to enzyme kinetics, mediator and substrate are both approximately at their bulk concentrations throughout the film in case I. The current is first order in both mediator and enzyme concentration and k, the enzyme reoxidation rate. It increases linearly with film thickness since there is no... 

Michaelis—Menten kinetics kinetics describing processes such as the majority of Enzyme-mediated reactions in which the initial reaction rate at low substrate concentrations is first order but at higher substrate concentrations becomes saturated and zero order. Can also apply to excretion for some compounds. 

Figure 2.21 Gluthathione S-trans-ferase enzyme-mediated reaction ultimately yielding a sulfonated metabolite (from Field and Thurman, 1996).

In general, Michaelis and Menten envisioned enzyme-mediated reactions as involving the following simple sequence ... 

Different forms of silicon dioxide have been used as supports for solid-phase organic synthesis. Silica gel is a rigid, insoluble material, which does not swell in organic solvents. Commercially available silica gel differs in particle size, pore size (typically 2-10 nm), and surface area (typically 200-800 m2/g). Like macroporous, highly cross-linked polystyrene, silica gel enables efficient and rapid transfer of solvents and reagents to its entire surface. Because the synthetic intermediates are only located on the surface of the support, enzyme-mediated reactions can be realized on silica [189,190], Silica gel is particularly well suited for continuous-flow synthesis because its volume stays constant and diffusion rates are high. 

Amidines and sulfonamides have also been used as linkers for primary or secondary aliphatic amines (Entries 4, 5, and 7, Table 3.23). These derivatives are stable under basic and acidic reaction conditions and can only be cleaved by strong nucleophiles. Phenylalanine amides can be hydrolyzed by treatment with certain enzymes (Entry 8, Table 3.23), and can therefore be used for linking amines to supports compatible with enzyme-mediated reactions (CPG, some polyacrylamides, macroporous polystyrene, etc.). 

Biotin enzymes are believed to function primarily in reversible carboxvlahon-decarboxylation reactions. For example, a biotin enzyme mediates the carboxylation of propionic acid to methylmalonic add, which is subsequently converted to succinic acid, a dtric acid cycle intermediate. A vitamin Bl2 coenzyme and coenzyme A are also essential to this overall reaction, again pointing out the interdependence of the B vitamin coenzymes. Another biotin enzyme-mediated reaction is the formation of malonyl-CoA by carboxylation of acetyl-CoA ( active acetate ). Malonyl-CoA is believed lo be a key intermediate in fatly add synthesis. 

Once one finds out which of two stereoheterotopic ligands or faces of a substrate is involved in an enzyme-catalyzed reaction, one is in a position to make a meaningful statement as to the location of the substrate in relation to the active site of the enzyme. While considerations of prostereoisomerism are thus useful in helping elucidate the enzyme-substrate relationship in the activated complex of an enzyme-mediated reaction, it must also be stressed that such considerations in themselves are insufficient to provide the complete picture and that they must necessarily be supplemented by many other techniques in enzyme chemistry. 

Part II of this book represents the bulk of the material on the analysis and modeling of biochemical systems. Concepts covered include biochemical reaction kinetics and kinetics of enzyme-mediated reactions simulation and analysis of biochemical systems including non-equilibrium open systems, metabolic networks, and phosphorylation cascades transport processes including membrane transport and electrophysiological systems. Part III covers the specialized topics of spatially distributed transport modeling and blood-tissue solute exchange, constraint-based analysis of large-scale biochemical networks, protein-protein interactions, and stochastic systems. 

Rate laws that are different from simple mass action often arise in chemical and biochemical applications. Important examples in biochemistry are enzyme and transporter mediated reactions where it is often assumed that a number of discrete steps are involved in converting substrates to products. The individual steps may be governed by mass action, but the overall steady state flux through an enzyme can take a more complex form. 

Net flux for nearly irreversible reactions is proportional to reverse flux In computational modeling of biochemical systems, the approximation that certain reactions are irreversible is often invoked. In this section, we explore the consequences of such an approximation, and show that the flux through nearly irreversible enzyme-mediated reactions is proportional to the reverse reaction flux. 

There is almost no biochemical reaction in a cell that is not catalyzed by an enzyme. (An enzyme is a specialized protein that increases the flux of a biochemical reaction by facilitating a mechanism [or mechanisms] for the reaction to proceed more rapidly than it would without the enzyme.) While the concept of an enzyme-mediated kinetic mechanism for a biochemical reaction was introduced in the previous chapter, this chapter explores the action of enzymes in greater detail than we have seen so far. Specifically, catalytic cycles associated with enzyme mechanisms are examined non-equilibrium steady state and transient kinetics of enzyme-mediated reactions are studied an asymptotic analysis of the fast and slow timescales of the Michaelis-Menten mechanism is presented and the concepts of cooperativity and hysteresis in enzyme kinetics are introduced. 

Analysis and modeling of biochemical systems - topics covered include enzyme-mediated reactions, metabolic networks, signaling systems, biological transport processes, and electrophysiological systems. 

Calvin cycle (aka Calvin-Benson Cycle or Carbon Fixation) Series of biochemical, enzyme-mediated reactions during which atmospheric carbon dioxide is reduced and incorporated into organic molecules, eventually some of this forms sugars. In eukaryotes, this occurs in the stroma of the chloroplast. 

As for most enzyme mediated reactions, increases in temperature often increase activity in a characteristic fashion. The response of an enzyme to temperature increases, often evaluated as the Qjq parameter (i.e., the increase in activity over a 10°C range), varies among and within enzyme systems and can show compensation. Staal et al. (2003) have evaluated Qjo responses for N2 fixation on short time scales in a range of cyanobacteria including Trkhodesmium, which exhibited a Qio of 1.12 for dark N2 fixation over the temperature range of 20—35°C, and a much... 

Many chemicals form reactive, electrophilic intermediates and free radicals during their metabolism in the body. These can be formed via enzyme-mediated reactions (many of which are oxidations) or from autoxidation of small molecules like flavins and thiols. These electrophilic intermediates covalently react with nucleophilic sites in the cell, including glutathione (GSH) and thiol-containing proteins, causing cellular... 

However, this does not apply to the special situation when (1) the enzyme is a synthetase which catalyzes the formation of a covalent bond between the metabolite (or antimetabolite) and a second substrate, and (2) the second substrate is available only in a limited amount. In this case, the antimetabolite competes with the metabolite not only for the enzyme but also for the second substrate, with which it will combine covalently to form an inert product. Although this enzyme-mediated reaction of the antimetabolite is reversible by the corresponding metabolite in a competitive manner, due to its potentially crucial metabolic effect, (i.e., the elimination of another, limiting metabolite which is required for the same reaction step of the metabolic pathway), this reaction per se could be responsible for the over-all inhibitory effect of the antimetabolite. That is, in such particular cases, the metabolic target of the inhibitory action of the antimetabolite may be an enzymic reaction step in which it actually plays the role of a substrate. One might think that this type of situation is a rather special and unusual one, as it may be indeed however, it so happens that the first descovered and still important class of classical and semi-classical antimetabolites, the sulfonamides, appears to act in this manner, as indicated by the results of a recent study8 (see Section 3.2.). 

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