Enzymes, Action

Enzymes frequently function as catalysts in very specific ways. In general, four types of behavior can be described. 

Absolute specificity. In this type of behavior, the enzyme catalyses a single reaction. 

FIGURE 6.1 The lock and key model for the enzyme-substrate complex. 

Group specificity. A reaction of only a single type of functional group is catalyzed by the enzyme. 

Linkage specificity. In this case, the enzyme makes a specific type of bond labile. 

It is generally believed that the catalytic reactions occur in atleast two steps  

Step 1 A molecule of enzyme(E) and a molecule of substrate(S) collide and react to form an intermediate called the enzyme-substrate complex (ES). 

Step 2 The decomposition of ES complex to give product(s) and the active enzyme 

The formation of an ES complex affords a lower activation energy. 

Fischer s Lock and Key Theory (Rigid Template Model) 

Adsorbed enzyme acts on its substrate. Crude cellulase contains three types of enzymatic action against cellulose CBHI, which acts along the glucose polymer 

The detailed kinetics of the individual cellulase components is a subject of research and will not be discussed here. However, some characterizations have been made by taking the cellulase complex as a whole. In this case, the behavior can be expressed as a function of the enzyme dosage by Eq. (2)  

For cellulase, typically 0.15 r 0.7. This fractional reaction order is characteristic of many effects by cellulase, not just production of glucose. The fractional reaction order indicates a diminishing return on increasing enzyme dosage. The relationship between extent of hydrolysis and reaction time is also expressed by a fractional exponent in time, which indicates a loss of enzyme effectiveness over time. 

In dimethyl sulfoxide, intermolecular hydrogen bonding of imidazole was demonstrable (Drey and Fruton, 1965). This was also true for 4,4 (5,5 )bis-imidazolylmethane (BIM). For imidazole (0.65M) bands were seen at 3440, 3178, 3030, 2930, 2840, 2690, and 2579 cm . Upon progressive dilution to 0.076M, the intensity of the band at 3178 cm decreased and that at 3440 cm increased. For BIM (0.21 M), bands were observed at 3440, 3165, 2940, 2858, 2700, and 2581 cm . Upon dilution to 0.016M 

Carbon dioxide in water absorbs at 2343.5 cm . The observed small shift for bound CO 2 was attributed to a solvent effect. The absence of a larger spectral shift showed that this substrate is not appreciably distorted upon binding by the enzyme. The data of Riepe and Wang (1968) indicated that at a pcoz of 1 and 25°C the active site of the enzyme was about 25% saturated with CO2. Consequently, the measurement of peak height from the difference infrared spectrum was much less reliable than the measurement of frequency for the bound COj. On the other hand, carbonic anhydrase can be completely saturated by azide at fairly low total con-concentration of the latter. Therefore, quantitative infrared measurements of the concentration of the enzyme-azide complex were more easily carried out. 

The binding constant of bovine carbonic anhydrase (BCA) for CO2 was estimated directly from the difference spectra taken at various CO2 pressures. The height of the difference absorption peak at 2341 cm was measured as a function of pcoi- Values of 1/[BCA-C02 complex] were plotted vs the corresponding values of l/Pco2 in Fig. 15.1. The approximate value of the dissociation constant, 

Azide ion, both free and bound to metal-ion complexes, also absorbs strongly in this range. A comparison between the infrared spectra of azide bound to diethylene-triamine Zn(II) and Co(II) complexes or to the cobalt-enzyme and the corresponding spectrum for the native enzyme showed that the azide ion is coordinate to the Zn(II) atom of the native enzyme. Examination of the difference spectra in the presence of both azide and CO2 showed that the bound azide sterically interferes with the binding of CO2 in the hydrophobic cavity adjacent to the Zn(ll). 

In 33 % inert protein solutions, the azide ion absorbed maximally at 2046 cm . When azide was added to a solution of carbonic anhydrase, an absorption band also appeared at 2094 cm as shown in spectrum 1 of Fig. 15.2. When an excess of ethox-zolamide was subsequently added to the above solution composed of azide plus enzyme, the absorption band at 2094 cm became completely suppressed, as shown in spectrum 2 of Fig. 15.2. Thus, ethoxzolamide and azide compete for binding by the enzyme. Spectrum 1 in Fig. 15.2 represents complete saturation of the enzyme with 

---

Averbukh I Sh, Blumenfeld L A, Kovarsky V A and Perelman N F 1986 A model of the mechanism of enzyme action in terms of protein conformational relaxation Blochim. Blophys. Acta. 873 290-6... 

Enzyme action is frequently accelerated or retarded by the presence of other substances both organic and inorganic. Such substances have been divided into three categories (a) co-enzymes, without which certain enzymes are unable to function (i) activators, and (c) inhibitors. 

Living systems contain thousands of different enzymes As we have seen all are structurally quite complex and no sweeping generalizations can be made to include all aspects of enzymic catalysis The case of carboxypeptidase A illustrates one mode of enzyme action the bringing together of reactants and catalytically active functions at the active site... 

Enzyme degradation of leaf protein may occur during emshing and separation from the fiber. The amino acids produced by this enzyme action are soluble in the juice and may be lost unless all of the juice is recovered. 

Currently, a-amino acids are prepared by several routes such as by the fermentation of glucose, by enzyme action on several substances and by the hydrolysis of proteins. Many methods for synthesising the polymers are known, of which the polymerisation of A -carboxyanhydrides is of particular interest, as it yield-products of high molecular weight (Figure 18.24). 

H. Dugas and C. Penney, Bioorganic Chemistry A Chemical Approach to Enzyme Action, 3rd ed., Springer-Veilag, New York, 1996. 

Lenore Michaelis and Maud L. Menten proposed a general theory of enzyme action in 1913 consistent with observed enzyme kinetics. Their theory was based on the assumption that the enzyme, E, and its substrate, S, associate reversibly to form an enzyme-substrate complex, ES ... 

Enzym-praparat, n. enzyme preparation, -wir-kung, /. enzyme action. 

Garansteig, m. the period during which enzyme action mounts to a maximum. 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Living cells contain thousands of different kinds of catalysts, each of which is necessary to life. Many of these catalysts are proteins called enzymes, large molecules with a slotlike active site, where reaction takes place (Fig. 13.39). The substrate, the molecule on which the enzyme acts, fits into the slot as a key fits into a lock (Fig. 13.40). However, unlike an ordinary lock, a protein molecule distorts slightly as the substrate molecule approaches, and its ability to undergo the correct distortion also determines whether the key will fit. This refinement of the original lock-and-key model is known as the induced-fit mechanism of enzyme action. 

FIGURE 13.40 In the lock-and-key model of enzyme action, the correct substrate is recognized by its ability to fit into the active site like a key into a lock. In a refinement of this model, the enzyme changes its shape slightly as the key enters. 

Biotransformation Conversion of a chemical into one or more products by a biological mechanism (predominantly by enzyme action). 

Site-directed mutagenesis, used to change residues suspected of being important in catalysis or substrate binding, provides insights into the mechanisms of enzyme action. 

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for study of the mechanism of enzyme action. Inhibitors can be classified based upon their site of action on the enzyme, on whether or not they chemically modify the enzyme, or on the kinetic parameters they influence. KineticaUy, we distinguish two classes of inhibitors based upon whether raising the substrate concentration does or does not overcome the inhibition. 

In addition to the catalytic action served by the snRNAs in the formation of mRNA, several other enzymatic functions have been attributed to RNA. Ribozymes are RNA molecules with catalytic activity. These generally involve transesterification reactions, and most are concerned with RNA metabofism (spfic-ing and endoribonuclease). Recently, a ribosomal RNA component was noted to hydrolyze an aminoacyl ester and thus to play a central role in peptide bond function (peptidyl transferases see Chapter 38). These observations, made in organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme. This has revolutionized thinking about enzyme action and the origin of life itself. 

---