Enzyme-catalyzed reactions features

The Qxo, or temperature coefficient, is the factor by which the rate of a biologic process increases for a 10 °C increase in temperature. For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10 °C rise in temperature (Qjo = 2). Changes in the rates of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for cold-blooded life forms such as lizards or fish, whose body temperatures are dictated by the external environment. However, for mammals and other homeothermic organisms, changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia. 

In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context. 

Virtually all biological reactions are stereospecific. This generalization applies not only to the enzyme-catalyzed reactions of intermediary metabolism, but also to the processes of nucleic acid synthesis and to the process of translation, in which the amino acids are linked in specific sequence to form the peptide chains of the enzymes. This review will be restricted mainly to some of the more elementary aspects of the stereospecificity of enzyme reactions, particularly to those features of chirality which have been worked out with the help of isotopes. 

Although conformational changes are essential features of proteins, the conformational basis of protein activity is not yet understood at the molecular and atomic levels. It is generally assumed that the mechanism of enzyme-catalyzed reactions would he defined if all the intermediates and transition states between the initial and final stages, as well as the rate constants, could be characterized. But in spite of constant progress in such characterization, most enzymatic mechanisms are not understood in terms of physical organic chemistry and enzyme activity is still regarded as a miracle as compared to classical catalysis. 

Burbaum et al. considered how kinetic/thermodynamic features of present-day enzyme-catalyzed reactions suggest that enzyme evolution tends to maximize catalytic effectiveness. They analyzed Uni Uni enzymes in terms of reaction energetics. Catalytically optimized enzymes... 

An enzyme circumvents these problems by providing a specific environment within which a given reaction can occur more rapidly. The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site (Fig. 6-1). The molecule that is bound in the active site and acted upon by the enzyme is called the substrate. The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. Often, the active site encloses a substrate, sequestering it completely from solution. The enzyme-... 

The transfer of phosphoryl groups is a central feature of metabolism. Equally important is another kind of transfer, electron transfer in oxidation-reduction reactions. These reactions involve the loss of electrons by one chemical species, which is thereby oxidized, and the gain of electrons by another, which is reduced. The flow of electrons in oxidation-reduction reactions is responsible, directly or indirectly, for all work done by living organisms. In nonphotosynthetic organisms, the sources of electrons are reduced compounds (foods) in photosynthetic organisms, the initial electron donor is a chemical species excited by the absorption of light. The path of electron flow in metabolism is complex. Electrons move from various metabolic intermediates to specialized electron carriers in enzyme-catalyzed reactions. 

Metabolic regulation, a central theme in biochemistry, is one of the most remarkable features of a living cell. Of the thousands of enzyme-catalyzed reactions that can take place in a cell, there is probably not one that escapes some form of regulation. Although it... 

Michaelis and Menten proposed a simple model that accounts for most of the features of enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently breaks down to product, regenerating the free enzyme. The model, involving one substrate molecule, is represented below ... 

In addition to cobalt and iron (discussed above), other metals frequently function as cofactors in enzyme-catalyzed reactions. Like coenzymes, they are useful because they offer something not available in amino acid side chains. The most important of such features of metals are their high concentration of positive charge, their directed valences for interacting with two or more ligands, and their ability to exist in two or more valence states. 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

Before we could take up this study in general, we had to solve one of the more bothersome aspects of cyclodextrin chemistry. It was believed strongly that cyclodextrin would bind substrates only in pure water solution, and this was a serious defect. First of all, it severely restricted the range of substrates that could be examined, since many interesting molecules have low water solubility. As a second point, it made it difficult to examine another feature of enzyme-catalyzed reactions. One of the roles that can be ascribed to the large protein mass, which contains the functional groups of an enzyme, is the function of water exclusion. That is, enzyme reactions can be considered to be operating in a nonaqueous or only partially aqueous medium. 

One source of nonlinear compartmental models is processes of enzyme-catalyzed reactions that occur in living cells. In such reactions, the reactant combines with an enzyme to form an enzyme-substrate complex, which can then break down to release the product of the reaction and free enzyme or can release the substrate unchanged as well as free enzyme. Traditional compartmental analysis cannot be applied to model enzymatic reactions, but the law of mass-balance allows us to obtain a set of differential equations describing mechanisms implied in such reactions. An important feature of such reactions is that the enzyme... 

One remarkable feature of enzyme-catalyzed reactions is the ability to generate at enzyme sites reactive intermediates, such as carbanions, carbocations, and radicals, that normally would require strong reaction conditions to generate in chemical reactions and would be very unstable in aqueous solution. 

Enzymes are high-molecular-weight protein molecules that catalyze reactions of importance in biology and biomedicine. Feature 29-1 discusses the basic features of enzymes. Enzymes are particularly useful as analytical reagents because of their selectivity. Consequently, they are widely used in the determination of molecules with which they combine when acting as catalysts. Such molecules are usually designated as substrates. In addition to the determination of substrates, enzyme-catalyzed reactions are employed for the determination of activators, inhibitors, and, of course, enzymes themselves. ... 

These interactions are frequently ionic in character. The coulombic forces of interaction between macroions and lower molecular weight ionic species are central to the life processes of the cell. For example, intermolecular interactions of nucleic acids with proteins and small ions, of proteins with anionic lipids and surfactants and with the ionic substrates of enzyme catalyzed reactions, and of ionic polysaccharides with a variety of inorganic cations are all improtant natural processes. Intramolecular coulombic interactions are also important for determining the shape and stability of biopolymer structures, the biological function of which frequently depends intimately on the conformational features of the molecule. 

Many of the observed attributes of enzymes arise by natural selection in order to help the host organism survive and reproduce. Benner et al. have proposed that one such attribute, the stereospecificities of dehydrogenases, has functional significance based on stereochemical arguments (18, 79). The central features of their functional model can be summarized as follows. The stereospecificities of dehydrogenases acting on alcohols are correlated with the equilibrium constant for the alcohol-carbonyl redox reaction as listed in Table IV (18). Enzymes catalyzing reactions where the eq is <10 " ilf transfer the pro-S proton from NADH when is >10"" Af, the pro-R proton is transferred. Thus the more readily reduced carbonyl compounds use the pro-R proton, but the more difficult to reduce carbonyl compounds use the pro-S proton. The proposed correlation is restricted to simple aldehydes and ketones (i.e., without additional chemistry that would influence the equilibrium constant, such as cyclizations of polyols or formation of lactones). The natural substrate of the enzyme must be well... 

How does enzyme-substrate binding result in a faster chemical reaction The precise answer to this question is probably different for each enz)une-substrate pair, and, indeed, we understand the exact mechanism of catalysis for very few enzymes. Nonetheless, we can look at the general features of enz)une-substrate interactions that result in enhanced reaction rate and product formation. To do this, we must once again look at the steps of an enzyme-catalyzed reaction, focusing on the remaining steps highlighted in blue ... 

The natural specificity of enzyme-catalyzed reactions can be used as the basis for selective detection of analytes (49, 64-68). One fruitful approach has featured potentiometric sensors with a structure similar to that of Figure 2.4.5, with the difference that the gap between the ion-selective electrode and the polymer diaphragm is filled with a matrix in which an enzyme is immobilized. 

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