Amino acid synthesis feedback inhibition/activation

A second class of herbicides primarily affects ( -carotene desaturase. These herbicides are apparent feedback inhibitors of PD as well. This class of compounds includes dihydropyrones like LS 80707 [90936-96-2] (56) and 6-methylpyridines (57,58). The third class consists of the ben2oylcyclohexane-diones, eg, 2-(4-chloro-2-nitroben2oyl)-5,5-dimethyl-cyclohexane-I,3-dione. This class of atypical bleaching herbicides induces phytoene accumulation when appHed either pre- or post-emergence. However, it does not inhibit phytoene desaturase activity in vitro (59). Amitrole also has been considered a bleaching herbicide, though its main mode of action is inhibition of amino acid synthesis. 

The role of the end products of a metabolic pathway in regulating their own biosynthesis was first demonstrated by Roberts et al. (1955). Working with E. co/z, they showed that amino acid synthesis from glucose is inhibited by the addition of amino acids to the incubation medium. Umbarger (1956) demonstrated that end products may inhibit the activity of enzymes mediating end-product synthesis. Often this inhibition is exerted on the first enzyme of the metabolic sequence. End products may also inhibit enzyme synthesis itself, as is frequently observed in anabolic pathways for amino acids, purines, and pyrimidines. This latter mode of metabolic regulation is termed repression and may occur independently of feedback inhibition. Both mechanisms may be involved in regulation of the same biosynthetic pathway. However, unlike feedback inhibition, which provides very rapid control, repression is a relatively slow process which permits adjustment of metabolism over an extended period of time. 

Catecholamine biosynthesis begins with the uptake of the amino acid tyrosine into the sympathetic neuronal cytoplasm, and conversion to DOPA by tyrosine hydroxylase. This enzyme is highly localized to the adrenal medulla, sympathetic nerves, and central adrenergic and dopaminergic nerves. Tyrosine hydroxylase activity is subject to feedback inhibition by its products DOPA, NE, and DA, and is the rate-limiting step in catecholamine synthesis the enzyme can be blocked by the competitive inhibitor a-methyl-/)-tyrosine (31). 

Feedback inhibition of amino acid transporters by amino acids synthesized by the cells might be responsible for the well known fact that blocking protein synthesis by cycloheximide in Saccharomyces cerevisiae inhibits the uptake of most amino acids [56]. Indeed, under these conditions, endogenous amino acids continue to accumulate. This situation, which precludes studying amino acid transport in yeast in the presence of inhibitors of protein synthesis, is very different from that observed in bacteria, where amino acid uptake is commonly measured in the presence of chloramphenicol in order to isolate the uptake process from further metabolism of accumulated substances. In yeast, when nitrogen starvation rather than cycloheximide is used to block protein synthesis, this leads to very high uptake activity. This fact supports the feedback inhibition interpretation of the observed cycloheximide effect. 

The affect of Li+ on the metabolism of serotonin (5-hydroxytryp-tamine, 5-HT) is equivocal. A number of studies consistently find a Li+-induced increase in the levels of the major metabolite, 5-hydroxyin-doleacetic acid (5-HIAA), in rat brain and in human CSF [155], which appears to reflect an increase in the rate of synthesis of 5-HT [156]. Li+-induced increases in the level of the amino acid precursor, tryptophan, and in the uptake of tryptophan by brain have also been reported [157], implying elevated tryptophan availability during Li+ treatment. In rat brain, chronic Li+ decreases the activity of tryptophan hydroxylase, the enzyme which, when activated by a Ca2+ and calmodulin-dependent protein kinase, leads to the synthesis of 5-HT [158]. Ca2+ increases the strength of binding of tryptophan to the enzyme, whereas Li+ has the opposite effect [159]. Tryptophan uptake is coupled to 5-HT utilization by a negative feedback mechanism and, therefore, the Li+-induced inhibition of tryptophan hydroxylase with a resultant decrease in 5-HT utilization could produce the observed increase in tryptophan uptake. 

Alkaloid metabolism in lupine was proved by Wink and Hartmann to be associated with chloroplasts (34). A series of enzymes involved in the biosynthesis of lupine alkaloids were localized in chloroplasts isolated from leaves of Lupinus polyphylls and seedlings of L. albus by differential centrifugation. They proposed a pathway for the biosynthesis of lupanine via conversion of exogenous 17-oxosparteine to lupanine with intact chloroplasts. The biosynthetic pathway of lupinine was also studied by Wink and Hartmann (35). Two enzymes involved in the biosynthesis of alkaloids, namely, lysine decarboxylase and 17-oxosparteine synthetase, were found in the chloroplast stoma. The activities of the two enzymes were as low as one-thousandth that of diaminopimelate decarboxylase, an enzyme involved in the biosynthetic pathway from lysine to diaminopimelate. It was suggested that these differences are not caused by substrate availability (e,g., lysine concentration) as a critical factor in the synthesis of alkaloids. Feedback inhibition would play a major role in the regulation of amino acid biosynthesis but not in the control of alkaloid formation. 

Biosynthetic pathways are often highly regulated such that blocks are synthesized only when supplies are low. Very often, a high concentration of the final product of a pathway inhibits the activity of enzymes that function early in the pathway. Often present are allosteric enzy capable ot sensing and responding to concentrations of regulatory species. These enzymes are similar in functional properties to aspartate transcar-bamylase and its regulators (Section 10.1). Feedback and allosteric mechanisms ensure that all 20 amino acids are maintained in sufficient amounts for protein synthesis and other processes. 

Feedback Inhibition and Activation. Two pathways with a common initial step may each be inhibited by its own product and activated by the product of the other pathway. Consider, for example, the biosynthesis of the amino acids valine, leucine, and isoleucine. A common intermediate, hydroxy ethyl thiamine pyrophosphate (hy-druxyethyl-TPP p, 478), initiates the pathways leading to all three of these amino acids. Hydroxyethyl-TPP reacts with a-ketobutyrate in the initial step for the synthesis of isoleucine. Alternatively, hydroxyethyl-TPP reacts with pyruvate in the committed step for the pathways leading to valine and leucine. Thus, the relative concentrations of a-ketobutyrate and pyruvate determine how much isoleucine is produced compared with valine and leucine. Threonine... 

The activity of enzymes can be regulated by a number of means. The enzymes involved in the synthesis and hydrolysis of glycogen can be activated by phosphorylation, and deactivated by dephosphorylation. This is an example of covalent modification. Amino acids are synthesized by sequences of up to 15 separate enzymatically catalyzed reactions. If the end product is present in high concentrations, it combines with the first enzyme in the synthetic sequence and shuts it down. This is an example of feedback inhibition. Other kinds of feedback inhibition will prevent the synthesis of the enzyme itself by interfering with the transcription step producing messenger RNA. 

Feedback repression is the inhibition of formation of one or more enzymes in a pathway by a derivative of the end product. In many (but not all) amino acid biosynthetic pathways, the amino add end product must first combine with its transfer RNA (tRNA) before it can cause repression. Feedback repression is a widespread regulatory device especially for the synthesis of molecules intended for incorporation into macromolecules, e.g. amino adds, purines, and pyrimidines. Synthesis of vitamins also appears to be controlled by feedback repression, as well as by catabolite regulation (Birnbaum et al, 1967 Sasaki, 1965 Newell and Tucker, 1966 Wilson and Pardee, 1962 Papiska and Lichstein, 1968). Regulation of vitamin synthesis is important since only a small number (probably about 1000) of vitamin molecules are required per cell whereas many molecules of an average amino acid (probably 50 million) are required. An extremely wasteful case of vitamin overproduction would develop if enzymes for vitamin synthesis were produced at the same rate and were as active as the amino acid biosynthetic enzymes. 

An excellent example of allosteric regulation—the control of an allosteric enzyme—is the five-step synthesis of the amino acid isoleucine (see I Figure 10.13). Threonine deaminase, the enzyme that catalyzes the first step in the conversion of threonine to isoleucine, is subject to inhibition by the final product, isoleucine. The structures of isoleucine and threonine are quite different, so isoleucine is not a competitive inhibitor. Also, the site to which isoleucine binds to the enzyme is different from the enzyme active site that binds to threonine. This second site, called the allosteric site, specifically recognizes isoleucine, whose presence there induces a change in the conformation of the enzyme such that threonine binds poorly to the active site. Thus, isoleucine exerts an inhibiting effect on the enzyme activity. As a result, the reaction slows as the concentration of isoleucine increases, and no excess isoleucine is produced. When the concentration of isoleucine falls to a low enough level, the enzyme becomes more active, and more isoleucine is synthesized. This type of allosteric regulation in which the enzyme that catalyzes the first step of a series of reactions is inhibited by the final product is called feedback inhibition. 

The synthesis of tryptophan in microorganisms is affected at several levels by end-product inhibition. Thus, end-product feedback inhibition partly regulates the synthesis of chorismic acid which is the final product of the common aromatic pathway and serves as a substrate for the first reaction in the tryptophan-synthesizing branch pathway (see Fig. 2). Regulation of the common aromatic pathway was recently reviewed by Doy [72]. The first enzyme of the common aromatic pathway, 3-deoxy-D-flrah/>jo-heptulosonate 7-phosphate synthetase (DAHPS), has been reported to exist as at least three isoenzymes, each specifically susceptible to inhibition by one of the aromatic amino acid end products (tyrosine, phenylalanine, and tryptophan), in E. coli (see reference [3]). It should be noted that many reports have indicated that in E. coli the DAHPS (trp), the isoenzyme whose synthesis is repressed specifically by tryptophan, was not sensitive to end-product inhibition by tryptophan. Recently, however, tryptophan inhibition of DAHPS (trp) activity has been demonstrated in E. coli [3,73,74]. The E. coli pattern, therefore, represents an example of enzyme multiplicity inhibition based on the inhibition specificity of isoenzymes. It is interesting to note the report by Wallace and Pittard [75] that even in the presence of an excess of all three aromatic amino acids enough chorismate is synthesized to provide for the synthesis of the aromatic vitamins whose individual pathways branch from this last common aromatic intermediate. In S. typhimurium, thus far, only two DAHPS isoenzymes, DAHPS (tyr) and DAHPS (phe) have been identified as sensitive to tyrosine and phenylalanine, respectively [76]. 

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