Citric acid enzymes from, inhibition

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

The toxicity of fluoroacetic acid and of its derivatives has played an historical decisive role at the conceptual level. Indeed, it demonstrates that a fluorinated analogue of a natural substrate could have an activity profile that is far different from that of the nonfluorinated parent compound. The toxicity of fluoroacetic acid is due to its ability to block the citric acid cycle (Krebs cycle), which is an essential process of the respiratory chain. The fluoroacetate is transformed in vivo into 2-fluorocitrate by the citrate synthase. It is generally admitted that aconitase (the enzyme that performs the following step of the Krebs cycle) is inhibited by 2-fluorocitrate the formation of aconitate through elimination of the water molecule is a priori impossible from this substrate analogue (Figure 7.1). 

Figure 11-3 Feedback inhibition of enzymes involved in the biosynthesis of threonine, isoleucine, methionine, and lysine in E. coli. These amino acids all arise from L-aspartate, which is formed from oxaloacetate generated by the biosynthetic reactions of the citric acid cycle (Fig. 10-6). Allosteric inhibition. Q Repression of transcription of the enzyme or of its synthesis on ribosomes.

The main reason is probably that the system evolved to keep the fumarate concentration low, because fumarate (and arginine) readily inhibits argininosuccinate lyase. Thus, this enzyme is cytoplasmic it is not inhibited by the high concentration of fumarate from the citric acid cycle since this fumarate is in the mitochondrion. 

As is the case with sorbic acid, benzoic acid penetrates the cell wall in the undissociated form. As a consequence, it is active at lower pH values only (pKa at 25°C = 4.19) and therefore serves as a preservative for sour products such as fruit juices and jams. In shrimp preservation it is applied as a powder that is spread over the shrimps, passes cell walls, and then ionizes in the intracellular fluid to yield protons that acidify the alkaline interior of the cell. The main cause of its activity, however, is biochemical effects (Eklund, 1980) such as inhibition of oxidative phosphorylation and of enzymes from the citric acid cycle (Chipley, 1983). In mayonnaise preserved by benzoic acid, the undissociated acid is mainly present in the lipid phase, which can be considered as a reservoir for the aqueous phase. 

Sorbic acid has a very low dissociation constant (1.73 x 10 , which means that it can be used also in food with relatively high pH-levels. The microbiostatic action of sorbic add can be explained mainly by its inhibitory action on different enzyme mechanisms. In particular enolase, lactatdehydrogenase and several other enzymes from the citric acid cycle and free sulfur-groups containing enzymes are inhibited in the cell (York and Vaughn, 1964 Rehm, 1967). Additionally sorbic acid acts also on the cell membrane (Eklund, 1981 and 1985 Stratford and Anslow, 1998). 

The results are of comparative interest but may have little bearing on the present discussion because the regulatory mechanisms for microbial fatty acid synthesis and for fatty acid synthesis in animal tissues appear to operate at quite different sites of control. Apart from the obvious absence of primary hormonal signals in bacteria, the following differences stand out. Only animal tissue acetyl-CoA carboxylase is activated by citric acid bacterial, plant and yeast carboxylase do not respond to this type of allosteric modulation. Similarly, microbial acetyl-CoA carboxylases are much more resistant to inhibition by palmitoyl-CoA at least at the concentration which inhibit the hepatic enzyme. 

The ability of an enzyme to respond to concentrations of metabolites other than its substrate and product adds a new dimension to metabolic regulation. It allows the end product of the metabolic pathway to bring about feedback inhibition on earlier steps (Yates and Pardee, 1956). Such feedback control may be exerted by a metabolite several steps removed in a pathway or by metabolites from a different pathway which share a common intermediate with the first pathway. This regul-ability in strategically located enzymes can have profound effects on cellular metabolism. It allows certain key intermediates in one pathway to act as switches for another pathway for example, citric acid can act as the switch for fatty acid metabolism. Regulation by central intermediates—e.g., adenine and pyridine nucleotides—may in fact determine the resultant direction of metabolism as anabolic or catabolic, depending on the energy reserves or redox state of the cell (see metabolite ratios below). 

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