1,4-addition active substrate control

Entries 7, 8, and 10 describe so-called Idnetically controlled syntheses starting from activated substrates such as ethyl esters or lactose. In two reaction systems it was possible to demonstrate that ionic liquids can also be useful in a thermodynamically controlled synthesis starting with the single components (Entry 11) [39]. In both cases, as with the results presented in entry 6, the ionic liquids were used with addition of less than 1 % water, necessary to maintain the enzyme activity. The yields observed were similar or better than those obtained with conventional organic solvents. 

Very few optically active cyanohydrins, derived from ketones, are described in the literature. High diastcrcosclectivity is observed for the substrate-controlled addition of hydrocyanic acid to 17-oxosteroids27 and for the addition of trimethyl(2-propenyl)silane to optically active acyl cyanides28. The enantioselective hydrolysis of racemic ketone cyanohydrin esters with yeast cells of Pichia miso occurs with only moderate chemical yields20. 

Inhibitor assay A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37°C. Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. 

This new system is an extension of the basic system discussed above. In the extended basic system an external inhibitor is also involved in the processes taking place. This component provides an additional path for control of the enzymic activity. Thus, whereas in the basic system the input signal is composed of concentration profiles of the consumable substrates, here the input signal contains a component that is an effector for one of the enzymes but is not consumed in the reactions. Due to this characteristic, this system is considered useful in terms of information processing only with continuous operational modes. 

Since preliminary studies showed that 6-hydroxymellein-O-methyl-transferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that S-adenosyl-Z.-homocysteine (SAH), which is a common product of many O-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-Omethyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the specific product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6-methoxymellein inhibits transferase activity with respect not only to 6-hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein-O-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6-methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should... 

In addition to structure control, metal ions can act as reactive centers of proteins or enzymes. The metals can not only bind reaction partners, their special reactivity can induce chemical reaction of the substrate. Very often different redox states of the metal ions play a crucial role in the specific chemistry of the metal. Non-redox-active enzymes, e.g. some hydrolytic enzymes, often react as a result of their Lewis-acid activity [2], Binding of substrates is, however, important not only for their chemical modification but also for their transport. Oxygen transport by hemoglobin is an important example of this [3]. 

Control of an electrolytic reaction often requires that the proton activity remains within acceptable limits during the electrolysis. For small-scale electrolytic preparations (less than about 10 g/liter of substrate), a sufficiently high initial concentration of buffer, acid, or base is adequate in aqueous solution for large-scale electrolysis a controlled addition of protons during a reduction must be provided. This addition may be controlled by a pH-stat or coupled to the current integrator. In aprotic media a proton donor, electrophile, or nucleophile may play a similar role as buffers in aqueous media. 

Bark of P. taeda obtained from a pulp mill in Mississippi was milled, extracted, activated, and reacted with bisaminopropylethylenediamine (BAPED). The modification was made to increase the capacity of the bark to adsorb anions. Modified bark (0.5 to 0.7 g) in 5 mL of 40 mM tartarate buffer pH 5.0 and 10 pL laccase was mixed for 30 min at room temperature. Phloroglucinol (1 mL of 0.3 M in ethanol) or catechol (1 mL of 0.3 M) were added and incubated for 3 h at room temperature, followed by 18 h at 5°C. The particles were rinsed and recovered by filtration and freeze dried. Controls included laccase addition without substrate and untreated modified bark. The bark was tested for the ability to remove phosphate from solution. 

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