Three-enzyme system

Cytochrome P4502E1, also microsomally located, catalyzes the hydroxylation of phenol to form hydroquinone (and to a much lesser extent catechol), which is then acted upon by the phase II enzymes (Benet et al. 1995 Campbell et al. 1987 Gut et al. 1996 McFadden et al. 1996). All three enzyme systems are found in multiple tissues and there is competition among them not only for phenol but for subsequent oxidative products, like hydroquinone. As a consequence, the relative amount of the products formed can vary based on species, dose and route of administration. In vivo, the gastrointestinal tract, liver, lung, and kidney appear to be the major sites of phenol sulfate and glucuronide conjugation of simple phenols (Cassidy and Houston 1984 Powell et al. 1974 Quebbemann and Anders 1973 ... 

Figure 5. Oxidation of methanol to carbon dioxide by a three-enzyme system consisting of alcohol (ADH), aldehyde (AldDH), and formate (FDH) dehydrogenases. Each enzyme is NAD+-dependent, and the NAD+ is regenerated by the anode via a redox mediator system. Redrawn with permission from ref 82. Copyright 1998 Elsevier Science S.A.

Fig. 5 Enzymatic oxidation of highly hydrolysed PVA proceeds in two steps 1,3-diol elements in PVA are oxidised via the [l-OH-ketone to form a diketone moiety. Three enzyme systems using different electron acceptors as cofactors/cosubstrates are shown...

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

An oxidative pentose phosphate cycle. Putting the three enzyme systems together, we can form a cycle that oxidizes hexose phosphates. Three carbon... 

Alternatively, ribulose (D-eryt/iro-pentulose) 5-phosphate may be iso-merized to ribose 5-phosphate with pentose phosphate isomerase, but the same isomerase will convert D-ribose 5-phosphate into D- ryt/zro-pentulose 5-phosphate, the equilibrium being displaced by phosphorylation to the diphosphate (involving three enzyme systems). 

Concerning the enzyme acted on the substrate, some researchers considered all three components of the cellulase enzyme system (endoglucanases, cellobiohydrolases, and cellobiase). Since the kinetic behavior of the three enzyme system are different and not fully understood, simplified models were also suggested by assuming the cellulase system can be represented quantitatively by a single enzyme. 

There are three enzyme systems involved in the metabolism of ethanol ... 

Deracemization by Stereoinversion via the Three-enzyme System i-Amino Acid Oxidase, D-Amino Transferase and Amino Add Racemase... 

The preparation of D-amino acids with the above three-enzyme system requires enzymes with opposite stereochemical selectivity and a suitable amino add as a donor. While D-amino add oxidase is an enzyme, the function of which in Nature is mainly related to the ehmination of D-amino adds, L-amino acid oxidases are usually found in aggressive animals (snakes). Bacterial L-amino acid oxidases often show a specific activity that is too low for preparative purposes [33]. Moreover, D-amino transferases are less common than the L-specific ones and require more expensive D-amino adds as amino donors. 

Benning C, Ohta, H. Three enzyme systems for galactoglyc-erolipid biosynthesis are coordinately regulated in plants. J. Biol. Chem. 2005 280 2397-2400. 

A total of three enzyme systems, which can be differentiated by column chromatography, are available in the liver cells for alcohol degradation. These enzyme systems differ with regard to hepatocellular localization and biochemical properties, (s. tab. 3.22)... 

Tab. 3.22 Localization-related and biochemical dififerences of the three enzyme systems responsible for alcohol degradation...

Our group has recently cloned a truncated Pd2,6ST containing 17-497 amino acid residues as N-hexohistine tagged protein and explored its application in the one-pot three-enzyme system for preparative synthesis of functionalized o2,6-sialosides (25). The tolerance of donor substrate modification by the purified Pd2,6ST was tested using the one-pot three-enzyme system, in which CMP-sialic acid derivatives were generated in situ from sialic acid precursors by the aldolase and NmCSS. An extremely relaxed donor substrate specificity was observed for Pd2,6ST. The preparative-sacle reactions were then carried out at... 

By controlling the pH and reaction time, PmSTl was used in preparative scale (20-150 mg) synthesis of a2,3-linked sialosides using the one-pot three-enzyme system similar to that described above for the Pd2,6ST. When the reaction was set up at the pH 7.0 to 9.0 for 1 to 2 h at 37 C, only a2,3-linked sialoside was formed and no significant sialidase or a2,6SiaT activitiy was observed under these conditions (24). 

Scheme 5. Chemoenzymatic synthesis of iGb3 using the three-enzyme system.

Over 90% of alcohol in the plasma is metabolized in the liver by three enzyme systems that operate within the hepatocyte. The remainder is excreted by the lungs and in urine and sweat. Alcohol is metabolized to acetaldehyde by alcohol dehydrogenase in the cell. In turn, acetaldehyde is metabolized to carbon dioxide and water by the enzyme aldehyde dehydrogenase. A second pathway for oxidation of alcohol uses catalase, an enzyme located in the peroxisomes and microsomes. The third enzyme system, the microsomal alcohol oxidase system, has a role in the oxidation of alcohol to acetaldehyde. These last two mechanisms are of lesser importance than the alcohol dehydrogenase-aldehyde dehydrogenase system. 

Three enzyme systems are required for the catabolism of sialic acids 0-acetylesterases, sialidases and lyases. Furthermore, a sialic acid transporter has been described (section 10.5), which carries liberated sialic acids from lysosomes into the cytosol, where they are either degraded by the lyase or recycled after activation with CTP (section 8.3). Sialic acid permeases provide bacteria with sialic acids for nutritional purposes (section 9.4). 

Complex xylulose structures can also be synthesized by RAMA1741. Employing a one-pot, three-enzyme system with RAMA, triose phosphate isomerase, and l-deoxy-D-xylulose-5-phosphate synthase, l-deoxy-D-xylulose-5-phosphate could be obtained in 47 % yield[75]. Furthermore, a four-enzyme, one-pot system employing FDP-aldolase from S. camosus furnished 5-deoxy-5-ethyl-D-xylulose1761. 

Specific target enzymes Based on the metabolic and catabolic pathways of limonin in Citrus, several enzymes have been identified for possible genetic manipulation. Specifically, we are currently looking at three enzyme systems 1) limonoate dehydrogenase, 2) limonin UDP-D-glucose transferase and 3) nomilin deacetylase. 

Presently, three enzyme systems have been identified as being capable of metabolizing organic nitrates, namely, a cytochrome P-450 (CYP) enzyme, a glutathione-S-transferase (GST), and a membrane-bound enzyme that has yet to be named. A recent review has addressed this topic (Bennett et al., 1994). Here, we examine the evidence, for and against, implicating each of these enzymes as the pharmacologically pertinent system. 

The above principle is amply illustrated by the small molecule systems discussed in Section III. The roles proposed for zinc ion in the three enzyme systems discussed in Section IV also adhere to this principle. The accumulated experimental evidence makes it highly probable that zinc ion has a Lewis acid catalytic function both in the horse liver alcohol dehydrogenase-catalyzed reduction of aldehydes, and in the carboxypeptidase A-catalyzed hydrolysis of peptides. In contrast, the accumulated experimental evidence supports a role for zinc ion involving the enhancement of water nucleophilicity via inner sphere coordination in the carbonic anhydrase-catalyzed hydration of CO 2. The substrates for... 

Equations (17) and (18) cannot be used directly for predictive purposes since they only describe and identify the components and not the original p/50 values directly. They clearly show, however, what the physical nature of the components is and in what direction stracture / has to be varied in order to arrive at more potent derivatives. True equations in the sense of a QSAR could have been obtained by the already mentioned special target rotation of Weiner and Malinowski, which would lead to a direct replacement of the abstract P j by molecule parameters according to equation (15). This, however, would not add new information in the present case, at least not for the three enzyme systems. For the ascites test the situation is a little different since here a small contribution of the first component in addition to the second component is present. If a Hansch analysis is directly applied to the experimental data of ascites inhibition alone a relation with % very similar to equation (18) is again obtained which can, however, be improved slightly by adding an electronic term. 

What distinguishes lethal synthesis from the cases of degradation before action discussed in Section 3.6 is simply that it is synthesis and not degradation a substance with only two carbon atoms has been made into one with six. Moreover, the raw material for this synthesis passes through at least three enzyme systems, and is changed a little by each, before it becomes toxic. 

---