Enzyme-mediated reactions

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). 

The rate of enzyme-mediated reactions, like most other types of reaction, depends on temperature. Over a limited temperature range, the reaction may follow the Arrhenius equation ... 

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... 

There are inherent problems associated with enzyme-mediated methods, regardless of the method used. The right conditions must be met, of course, for the enzyme action to take place. Unlike fluorochromes or gold particles (two other marker compounds), enzymes need to act chemically for the assay to work. Also, the enzyme action must only represent the marker molecule. Endogenous enzyme or enzyme-like activity can create problems only realized in systems that use enzymes. Also, the use of enzymes demands more attention to detail because of the increase in sensitivity that is often obtained. The problem of unwanted reactivity is enhanced in enzyme-mediated reactions more so than in others, in part because of the additional level of sensitivity brought about by the continuous action on a substrate. 

In this chapter, it has been demonstrated that DCR is an effective method for effective kinetic screening of dynamic libraries. By combining DCLs with enzyme-mediated reactions, complete resolution of the libraries could be achieved in a one-pot process. This approach also enables screening of complex DCLs without the necessity of equimolar amounts of target molecules. 

A cyclical depiction of an enzyme-mediated reaction written to account for the regenerative nature of catalytic processes. The reaction cycle for a typical one-substrate one-product enzyme mechanism may be written as follows ... 

Such enzyme-mediated reactions can produce different reactive intermediates, namely,... 

Is glutathione conjugation the result of a chemical reaction or an enzyme-mediated reaction ... 

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. 

Glutathione conjugation can be the result of either an enzyme-mediated reaction or a chemical reaction. 

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 ... 

The fact that macroporous, highly cross-linked polystyrene does not swell makes this support particularly interesting for continuous-flow synthesis in columns. This support has also been successfully used as an alternative to CPG for the solid-phase synthesis of oligonucleotides [90,91]. Furthermore, because reagents do not need to penetrate into the polystyrene network, enzyme-mediated reactions should also proceed smoothly on macroporous polystyrene [85]. 

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. 

Essential oil from nutmeg suppressed the formation of DNA adducts by afla-toxin B1 in vitro in a microsomal enzyme-mediated reaction (Hashim et al., 1994). A diet containing 1% mace inhibited the DMBA-induced papillomagenesis in the skin of male Swiss albino mice (Jannu et al., 1991). 

DDT-dehydrochlorinase, a reduced glutathione (GSH)-dependent enzyme, has been isolated from the 100,000g supernatant of resistant houseflies. Although the enzyme-mediated reaction requires glutathione, the glutathione levels are not altered at the end of the reaction (Figure 10.12). 

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. 

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. 

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