Soluble-enzyme complexes

Biosynthesis. The asymmetric incorporation of 4-(2 -carboxyphenyl-4-oxobu-tyrate [o-succinylbenzoate (211)] into phylloquinone by Zea mays has been reported. The incorporation of (211), via its coenzyme A thioester, into l,4-dihydroxy-2-naphthoic acid (212) and menaquinone has been studied in cell-free extracts of Mycobacterium phlei and Micrococcus luteus. The biosynthesis and metabolism of menaquinone-4 in the crab has been described. A soluble enzyme complex capable of converting 2-octaprenylphenol (213) into... 

Kim SJ, Kim MD, Choi JH, Kim SY, Ryu YW, Seo JH (2006) Amplification of 1-deoxy-D-xylu-ose 5-phosphate (DXP) synthase level increases coenzyme QIO production in recombinant Escherichia coli. Appl Microbiol Biotechnol 72 982-985 Knoell HE (1979) Isolation of a soluble enzyme complex comprising the ubiquinone-8 synthesis apparatus from the cytoplasmic membrane of Escherichia coli. Biochem Biophys Res Commun 91 919-925... 

As research reveals the ultrastructural organization of the cell in ever greater detail, more and more of the so-called soluble enzyme systems are found to be physically united into functional complexes. Thus, in many (perhaps all) metabolic pathways, the consecutively acting enzymes are associated into stable multienzyme complexes that are sometimes referred to as metabolons, a word meaning units of metabolism. ... 

FIGURE 18.5 Schematic representation of types of multienzyme systems carrying out a metabolic pathway (a) Physically separate, soluble enzymes with diffusing intermediates, (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes Ei to E5 before product is released. No intermediates are free to diffuse away, (c) A membrane-bound multienzyme system. 

The preparations of luciferin (Ln, an electron acceptor) and soluble enzyme used were crude or only partially purified. The luciferase was an insoluble particulate material, possibly composed of many substances having various functions. Moreover, the luciferin-luciferase reaction was negative when both luciferin and luciferase were prepared from certain species of luminous fungus. It appears that the light production reported was the result of a complex mechanism involving unknown substances in the test mixture, and probably the crucial step of the light-emitting reaction is not represented by the above schemes. 

For a soluble enzyme that is not part of a multi-enzyme complex, the fastest rate of enzyme-inhibitor association is determined by the rate of molecular collisions between the two binding partners (i.e., the enzyme and the inhibitor) in solution. The rate of molecular collisions is in turn controlled by the rate of diffusion. The diffusion-limited rate of molecular collisions is dependent on the radii of the two binding molecules and the solution temperature and viscosity (Fersht, 1999) ... 

For the first group (i.e. intracellular soluble enzymes and proteins), which need no posttranslational modification and complex domain organization influencing protein folding, E. coli is the most preferred choice. However, for the other targets, alternative expression systems often provide a higher rate of success. The most common expression systems are presented in this chapter. 

Enzyme membrane reactor for production of diltiazem intermediate. A solution of the racemic ester in organic solvent enters the port at the bottom of the reactor and flows past the strands of microporous, hollow-fiber membrane that contain an enzyme. The enzyme catalyzes hydrolysis of one enantiomer of the ester that undergoes decarboxylation to 4-methoxyphenylacetaldehyde (which in turn forms a water-soluble bisulfite complex that remains in the aqueous phase). The other enantiomer of the ester remains in the aqueous stream that leaves the reactor via the port at the top. Courtesy of Sepracor, Inc. 

Whereas antigen-retrieval technique serves to amplifying the immunocytochemical signal at the predetection phase, conventional methods of signal amplification, such as avidin biotin complex (ABC) and soluble enzyme-anti-enzyme immune complex techniques (peroxidase-anti-peroxidase complex and alkaline phosphatase-anti-alkaline phosphatase complex PAP and APAAP respectively), are applied in the phase of detection. For many years, the PAP and APAAP procedures represented the most sensitive and reliable and hence most popular techniques in many pathology laboratories. However, today these techniques are only rarely used, being substituted by modem more sensitive methods. 

Chelating agents are widely used as specific antidotes for heavy metals. They form stable, soluble, nontoxic complexes and in easily excreted form. They promote dissociation of bound metal from tissue enzymes and other functional macromolecules. These metal chelates are water soluble, e.g. EDTA, BAL, desferrioxamine etc. 

Cytochrome c is not part of an enzyme complex it moves between Complexes III and IV as a freely soluble protein. 

Generally, the assimilatory nitrate and nitrite reductases are soluble enzymes that utilize reduced pyridine nucleotides or reduced ferrodoxin. In contrast, the dissimilatory nitrate reductases are membrane-bound terminal electron acceptors that are tightly linked to cytochrome by pigments. Such complexes allow one or more sites of energy conservation (ATP generation) coupled with electron transport. 

Lead is widely destributed in the environment, especially in industrial and urban areas, and it is readily absorbed into the mammalian body where it exerts a number of undesirable physiological effects. Its most dramatic action is the inhibition of human red cell 5-aminolaevulinic acid dehydrase activity71), but it also depresses the activities of many enzymes having functionsl -SH groups. Attempts to remove lead from the body using agents such as dimercaptopropanol can result in the formation of lipid-soluble lead complexes that may be carried to the brain and exacerbate the effects of lead poisoning. 

Orange PE was shown to be bound to cell walls as an enzyme-substrate complex with pectin (24). The solubilization of PE (pH 7.5 and 0.15N NaCl) and de-esterification of cell wall pectin were similarly temperature dependent. After complete de-esterification of the cell wall pectin, an equilibrium was established between bound and free PE in the extraction medium. At the pH of juice (4.5 and below) the enzyme bound to cell walls was not solubilized unless soluble pectin was added. The bound enzyme was inactive at pH 4, whereas the free soluble enzyme was about 20% more active at pH 4 than at the optimum pH (7.5). 

The potent oxidizing properties of this activated oxygen permit oxidation of a large number of substrates. Substrate specificity is very low for this enzyme complex. High solubility in lipids is the only common structural feature of the wide variety of structurally unrelated drugs and chemicals that serve as substrates in this system (Table 4-1). 

Commercial yeast invertase (Bioinvert ) was immobilized by adsorption on anion-exchange resins, collectively named Dowex (1x8 50-400,1x4 50-400, and 1x2 100-400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/inver-tase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL. 

At 55°C, k was 0.006 (Eq. 8) and 0.0292 min-1 (Eq. 12), respectively, for soluble and insoluble invertase. As can be seen, the activity of Dowex-lx4-200/invertase complex diminished at a rate fivefold higher than the soluble enzyme, as the time increased. One possible explanation could be related to the probable different patterns of internal heat transference presented by a solution and a suspension. In a suspension, a significant portion of the thermal energy would accumulate in the resin particles in which the invertase molecules are adsorbed. As a consequence, the molecules should be submitted to a higher thermal energy than when they are homogeneously dispersed in a solution. 

Branched cydodextrins are also used to increase the solubility of complexes. Two methods are used to make branched cydodextrins, an enzymic method and a pyrolytic method. In the enzymic method, a starch debranching enzyme, such as pul-lulanase, is added to a solution of cyclodextrin and a large excess of D-glucose or maltose to force the reaction to proceed in the reverse direction, i.e. to add rather than remove a branch.69 Since the equilibrium favors the debranching reaction, yields are low and the product typically contains —15% branched cyclodextrin and —85% glucose or maltose. Purification is difficult because of the high solubility of both the glucose or maltose and the branched cyclodextrin, but much of the unreacted cyclodextrin can be removed by crystallization. 

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