Catalytic activity bound enzymes

The identification of the amino acids at the active center is very important if the mechanism of the enzymic reaction is to be understood, but unambiguous evidence concerning the nature of such acids is extremely diflScult to obtain. (It should be emphasized that the amino acids of the active site need not be close to one another in the amino acid sequence of the protein, as folding of the polypeptide chains can bring them together.) For catalytic activity, many enzymes require a non-protein ion or molecule, bound to (or in close association with) the enzyme—protein. The nature of this prosthetic group, or coenzyme, must be investigated in order that the enzymic action may be fully understood. 

Enzymes are important catalysts in biological organisms and are of increasing use in detergents and sensors. It is of interest to understand not only their adsorption characteristics but also their catalytic activity on the surface. The interplay between adsorption and deactivation has been clearly illustrated [119] as has the ability of a protein to cleave a surface-bound substrate [120]. 

Inspired by the many hydrolytically-active metallo enzymes encountered in nature, extensive studies have been performed on so-called metallo micelles. These investigations usually focus on mixed micelles of a common surfactant together with a special chelating surfactant that exhibits a high affinity for transition-metal ions. These aggregates can have remarkable catalytic effects on the hydrolysis of activated carboxylic acid esters, phosphate esters and amides. In these reactions the exact role of the metal ion is not clear and may vary from one system to another. However, there are strong indications that the major function of the metal ion is the coordination of hydroxide anion in the Stem region of the micelle where it is in the proximity of the micelle-bound substrate. The first report of catalysis of a hydrolysis reaction by me tall omi cell es stems from 1978. In the years that... 

Aqueous solutions are not suitable solvents for esterifications and transesterifications, and these reactions are carried out in organic solvents of low polarity [9-12]. However, enzymes are surrounded by a hydration shell or bound water that is required for the retention of structure and catalytic activity [13]. Polar hydrophilic solvents such as DMF, DMSO, acetone, and alcohols (log P<0, where P is the partition coefficient between octanol and water) are incompatible and lead to rapid denaturation. Common solvents for esterifications and transesterifications include alkanes (hexane/log P=3.5), aromatics (toluene/2.5, benzene/2), haloalkanes (CHCI3/2, CH2CI2/I.4), and ethers (diisopropyl ether/1.9, terf-butylmethyl ether/ 0.94, diethyl ether/0.85). Exceptionally stable enzymes such as Candida antarctica lipase B (CAL-B) have been used in more polar solvents (tetrahydrofuran/0.49, acetonitrile/—0.33). Room-temperature ionic liquids [14—17] and supercritical fluids [18] are also good media for a wide range of biotransformations. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

In the Kohn-Sham Hamiltonian, the SVWN exchange-correlation functional was used. Equation 4.12 was applied to calculate the electron density of folate, dihydrofolate, and NADPH (reduced nicotinamide adenine dinucleotide phosphate) bound to the enzyme— dihydrofolate reductase. For each investigated molecule, the electron density was compared with that of the isolated molecule (i.e., with VcKt = 0). A very strong polarizing effect of the enzyme electric field was seen. The largest deformations of the bound molecule s electron density were localized. The calculations for folate and dihydrofolate helped to rationalize the role of some ionizable groups in the catalytic activity of this enzyme. The results are,... 

All isoforms of PKC are predominantly localized to the cytosol and, upon activation, undergo translocation to either plasma or nuclear membranes. However, newly synthesized PKCs are localized to the plasmalemma and are in an open conformation in which the auto inhibitory pseudosubstrate sequence is removed from the substrate binding domain. The maturation of PKC isoforms is effected by phosphoinositide-dependentkinase-I (PDK-I), which phosphorylates a conserved threonine residue in the activation loop of the catalytic (C4) domain [24]. This in turn permits the autophosphorylation of C-terminus threonine and serine residues in PKC, a step which is a prerequisite for catalytic activity (see also Chs 22 and 23). The phosphorylated enzyme is then released into the cytosol, where it is maintained in an inactive conformation by the bound pseudosubstrate. It was originally thought that 3-phosphoinositides such as PI(3,4)P2 and PI(3,4,5)P3 could directly activate PKCs. However, it now seems more likely that these lipids serve to activate PDK-1 (a frequent contaminant of PKC preparations). 

Biochemical and molecular cloning studies indicate the existence of nine separate and unique forms of adenylyl cyclase (AC), which comprise a distinct enzyme family, referred to as AC1-AC9 [1, 2]. These members of the adenylyl cyclase superfamily are all membrane-bound. There is also an additional soluble form, sAC, that has catalytic activity similar to the others but is genetically the most divergent member of the family. All the membrane-bound forms of adenylyl cyclase are activated by the stimulatory G protein Gas (see Ch. 19), and all with the exception of AC9 are stimulated by forskolin. The soluble form sAC is not stimulated by either Gas or forskolin but is sensitive to bicarbonate levels. All known forms of... 

It is worth mentioning that membrane-bound forms of GC, which can be considered signal transducing enzymes , are structurally homologous to other signal transducing enzymes, such as certain protein tyrosine kinases and phosphatases, which also possess receptor moieties in their extracellular (amino terminus) domain and enzyme catalytic activity in their intracellular domain (see Ch. 24). Activation of many of these receptors occurs upon ligand-induced dimerization of the receptors, and a similar... 

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

Figure 6.3. Mechanism of action of heterotrimeric G-proteins. Upon receptor occupancy, the Ga-subunit binds GTP in exchange for GDP, and then moves in the membrane until it encounters its target enzyme, shown here as adenylate cyclase (alternatively, a phospholipase). The activated target enzyme then becomes functional. Inherent GTPase activity within the a-subunit then hydrolyses bound GTP to GDP, and the a-subunit dissociates from its target enzyme (which becomes inactive) and rebinds the / - and ysubunits. Upon continued receptor occupancy, further catalytic cycles of GTP exchange and target enzyme activation may occur. The scheme shown is for a stimulatory G-protein (Got,), but similar sequences of events occur with inhibitory G-proteins (Gcx,) except that the interaction of the a-subunit with adenylate cyclase will result in its inhibition. The sites of action of pertussis and cholera toxins are shown.

In addition, it has been shown that other enzymes such as trypsin can be successfully immobilized and used for the conversion of substrates with higher molecular masses [76]. Petro et al. [94] compared the activity of trypsin immobilized on macroporous beads and on monolithic supports. They were able to show that the catalytic activity of trypsin bound to a monolith was much higher and resulted in a much higher throughput. Other enzymes such as invertase [76] and... 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Figure 9.15 Enzymes in aqueous (light-coloured) and hydrophobic (shaded) phases. (A) A protein in the periplasm (PP) of a cell (OM = outer membrane, CM = cytoplasmic membrane) (B) membrane-bound protein in a lipid bilayer (C) hydrophilic protein in an inverted micelle (D) interaction between enzyme and substrates in aqueous micelles (E) graph of catalytic activity as a function of micelle concentration.

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