Enzyme-catalyzed reactions, long

Cyclodextrins as catalysts and enzyme models It has long been known that cyclodextrins may act as elementary models for the catalytic behaviour of enzymes (Breslow, 1971). These hosts, with the assistance of their hydroxyl functions, may exhibit guest specificity, competitive inhibition, and Michaelis-Menten-type kinetics. All these are characteristics of enzyme-catalyzed reactions. 

Suppose that the reaction between A and B to give the intermediate is very fast and very favorable. If we have more B than A to start with, all the A is converted instantly into the intermediate. If we re following P, what we observe is the formation of P from the intermediate with the rate constant k2. If we increase the amount of B, the rate of P formation won t increase as long as there is enough B around to rapidly convert all the A to the intermediate. In this situation, the velocity of P formation is independent of how much B is present. The reaction is zero-order with respect to the concentration of B. This is a special case. Not all reactions that go by this simple mechanism are zero-order in B. It depends on the relative magnitudes of the individual rate constants. At a saturating concentration of substrate, many enzyme-catalyzed reactions are zero-order in substrate concentration however, they are still first-order in enzyme concentration (see Chap. 8). 

Aconitase was first described 50 years ago by Martius (1,2) and soon there after named by Breusch (3). The enzyme demonstrated the then surprizing ability to distinguish between the chemicadly identical acetyl arms of citrate (4). The stereo-specificity of enzyme catalyzed reactions was not fully understood until the late 1940 s when Ogston point out that as long as a substrate attaches to an asymmetric enzyme at three points, the enzyme can differentiate between two identical amis of a symmetrical molecule (5). 

As noted earlier, the velocity of any enzyme-catalyzed reaction is dependent upon the amount of effective enzyme present. Enzyme biosynthesis, like that of all proteins, is under genetic control, a long-term process. Biosynthesis of enzymes may be increased or decreased at the genome level. Various hormones can activate or repress the mechanisms controlling gene expression. Enzyme levels are the result of the balance between synthesis and degradation. This enzyme turnover may be altered by diverse physiological conditions, by hormone effects, and by the level of metabolites. 

Mechanistic knowledge of enzyme-catalyzed reactions is being applied increasingly to incorporate minimalistic structural units to synthetic analogues. Some of the efforts described above have successfully recreated at least a fraction of the activities of natural enzymes. This biomimetic approach will be sustained as long as there is a need for developing stable, inexpensive catalysts that can survive the conditions in which the natural enzymes get denatured and do not sustain their activity. 

One example of an enzyme-catalyzed reaction involving a radical intermediate is the enzyme ribonucleotide reductase, which catalyzes the conversion of ribonucleotides (used for RNA biosynthesis) to 2 -deoxyribonucleotides (used for DNA biosynthesis), as illustrated in Fig. 16. Spectroscopic studies of the R2 subunit of Escherichia coli ribonucleotide reductase have shown that it can form a stable, long-lived, tyrosyl radical species—the first protein radical to be discovered (13). 

With enzyme determinations, standards for calibration purposes and for checking of instrumental performance make use of separately prepared solutions of one of the reactants or the products of the enzyme-catalyzed reaction. For instance, a solution of phenol may be standardized and thereafter used itself as the standard in determinations of acid and alkaline phosphatase, in methods employing phenyl phosphate as substrate and depending on the measurement of the amount of phenol liberated. This standard phenol solution, however, cannot be taken through all the steps in the determination of phosphatase, and a separate control solution must be used to check the performance of the overall technique if this control were omitted, it would be possible, for instance, for a buffer to be incorrectly prepared and for erroneous levels of phosphatase activity to be found. There is no substitute, therefore, in the control of enzyme determinations for the inclusion of a sample (of serum, urine, etc.) previously investigated for its level of enzyme activity. For long-term monitoring of an enzyme method, the repeated analysis of aliquots of a standardized enzyme preparation is most useful, provided... 

A reasonable ambition for model reactions is that their mechanisms ought to contain some dues about the mechanism of the enzyme-catalyzed reaction also. It has long been realized that it is fruitless simply to buUd the model-reaction mechanism into an enzyme active site. Such a procedure would entail the view that the factors present and at work in the model system render a complete account of the biological history of the enzyme. There is no reason to expect this to be so, and many reasons to think it would not be so. In the simplest sense, a given enzyme must occupy a niche in a metabolic network that may require its regulation and may influence its structure and mechanistic potentialities in ways that cannot be derived from non-enzymic studies. 

The dependence of an enzyme-catalyzed reaction rate on substrate concentration is illustrated in Figure 22.2. An enzyme is characterized by the number of molecules of substrate it can complex per unit time and convert to product, that is, the turnover number. As long as the substrate concentration is small enough with respect to the enzyme concentration that the turnover number is not exceeded, the reaction rate is directly proportional to substrate concentration, that is, it is first order with respect to substrate (Equation 22.13). If the enzyme concentration is held constant, then the overall reaction is first order and directly proportional to substrate concentration (k[E] = constant in Equation 22.13). This serves as the basis for substrate determination. However, if the amount of substrate exceeds the turnover number for the amount of enzyme present, the enzyme becomes saturated with respect to the number of molecules it can complex (saturated with respect to substrate), and the reaction rate reaches a maximum value. At this point, the reaction becomes independent of further substrate concentration increases, that is, becomes pseudp zeroj5r if the enzyme concentration is constant (Figure 22.2) in Equation 22.13, [ES] becomes constant and R = constant. 

Hydrophobic molecules can also denature proteins by disturbing hydrophobic interactions in the protein. For example, long-chain fatty acids can inhibit many enzyme-catalyzed reactions by binding nonspecifically to hydrophobic pockets in proteins and disrupting hydrophobic interactions. Thus, long-chain fatty acids and other highly hydrophobic molecules have their own binding proteins in the cell. 

When transcription occurs in the nuclei of eukaryotic cells, both introns and exons are transcribed. This produces what is called heterogeneous nuclear RNA (hnRNA). This long molecule of hnRNA then undergoes a series of enzyme-catalyzed reactions that cut and splice the hnRNA to produce mRNA (see > Figure 11.18). The mRNA resulting from this process contains only the sequence of bases that actually codes for protein synthesis. Although the function of introns in eukaryotic DNA is not yet understood, it is an area of active research. 

Figure 1 illustrates the dependence of an enzyme-catalyzed reaction rate on the substrate concentration. An enzyme is characterized by the number of molecules of substrate that it can convert to the product that is, turnover number. As long as the substrate concentration is small enough with respect to the enzyme concentration that the turnover number is not exceeded, the reaction rate is directly proportional to the substrate concentration that is, it is first order with respect to substrate. If the enzyme concentration is held constant, then the overall... 

The high sensitivity of fluorescence spectroscopy and the selectivity of enzymatic assays are responsible for the increasing use of fluorimetric methods in enzymology. Enzyme determinations usually involve the use of kinetic methodology for measuring the rate of formation of the fluorescent product, while both equilibrium and kinetic methods are used to determine the substrates. Fluorimetric measurements on enzyme-catalyzed reactions have been used for a long time to determine a variety of enzymes and substrates (Figure 2). 

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