Aspartokinase inhibition

Paulus, H. Gray, E. Multivalent feedback inhibition of aspartokinase in Bacillus polymyxa. I. Kinetic studies. J. Biol. Chem., 242, 4980-4986 (1967)... 

Dungan, S.M. Datta, P. Concerted feedback inhibition. Purification and some properties of aspartokinase from Pseudomonas fluorescens. J. Biol. Chem., 248, 8534-8540 (1973)... 

Dawson Funkhouser, J. Abraham, A. Smith, V.A. Smith W.G. Kinetic and molecular properties of lysine-sensitive aspartokinase. Factors influencing the lysine-mediated association reaction and their relationship to the cooperativity of lysine inhibition. J. Biol. Chem., 249, 5478-5484 (1974)... 

In many cases the amino acid pathway branches so that two or more amino acids are formed. Aspartate is the precursor of four other amino acids found in proteins Isoleucine, threonine, methionine, and lysine (see fig. 21.2). The first step in this overall pathway entails the conversion of aspartate to /3-aspartyl-phosphate by aspartokinase. One might imagine that all four of the amino acid end products of this pathway would act together to inhibit this enzyme. However, in E. coli a different solution has been found. In this bacterium there are three aspartokinases which appear to be parts of different multienzyme complexes leading to threonine and leucine for aspartokinase I, methionine for aspartokinase II and lysine for aspartokinase III. As might be expected threonine and isoleucine inhibit aspartokinase I,... 

S-(2-Aminoethyl)-L-cysteine (AEC), H2N-CH2-CH2-S-CH2-CH(NH2)-COOH, a lysine analog, acts as a false feedback inhibitor on aspartokinase, which produces aspartylphosphate from aspartate. The inhibitor simulates, for aspartokinase, the absence of lysine and threonine, and as a consequence the AEC insensitive mutant is no longer inhibited by lysine and threonine. The result was a yield increase from 0 to 16 g L 1. 

Overproduction of E (isoleucine) inhibits enzyme E6 (threonine deaminase), and the consequent rise of D (threonine) reduces the rate of production of C (homoserine) via enzyme E3 (homoserine dehydrogenase). The concentration of B (aspartate semialdehyde) rises, and this in turn inhibits Ej (aspartokinase). It is therefore obvious why the control system is called a negative feedback network, or sequential feedback system. 

Removal of negative regulations can also enhance L-threonine production. By removing L-lysine-mediated feedback inhibition of aspartokinase III encoded by the lysC gene, L-threonine production could be increased by 30.9% (11.0-14.4 g L 1) in E. coli mutant strain [57]. 

Ogawa-Miyata Y, Kojima H, Sano K (2001) Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production. Biosci Biotech Biochem 65 1149-1154... 

Sophisticated regulation can also evolve by duplication of the genes encoding the biosynthetic enzymes. For example, the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three distinct aspartokinases catalyze this reaction in E. coli, an example of a regulatory mechanism called enzyme multiplicity. (Figure 24.24). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the mechanisms of catalysis are essentially identical, their activities are regulated differently one enzyme is not subject to feedback inhibition, another is inhibited by threonine, and the third is inhibited by lysine. 

The pathway of biosynthesis of L-lysine and L-threonine in Corynebacterium glutamicum is shown in Fig. 1. The first step, the formation of phosphoaspartate from aspartate, is catalyzed by aspertokinase and this enzyme is susceptible to the concerted feedback inhibition by L-lysine and L-threonine. The auxotrophic mutant of homoserine (or threonine plus methionine), lacking homoserine dehydrogenase, was constructed and found to produce L-lysine in the culture medium. Second, the mutants which show the threonine or methionine sensitive phenotype caused by the mutation on homoserine dehydrogenase (low activity) was also found to produce appreciable amounts of L-lysine in the culture medium. Furthermore, a lysine analogue (S-aminoethylcysteine) resistant mutant was obtained as an L-lysine producer and in this strain aspartokinase was insensitive to the feedback inhibition. 

Aspartokinase is a major point of regulation of the biosynthetic pathways leading to threonine, lysine, and methionine. In bacteria, there are three isoenzymes of aspartokinase. Activity of one form is inhibited specifically by threonine and that of another form by lysine. Synthesis of the third form is inhibited by methionine. 

In E. coli there are three aspartokinases that catalyze the conversion of aspartate to p-aspartyl phosphate. All three catalyze the same reaction, but they have very different regulatory properties, as is indicated in Fig. 24-13. Each enzyme is responsive to a different set of end products. The same is true for the two aspartate semialdehyde reductases which catalyze the third step. Both repression of transcription and feedback inhibition of the enzymes are involved. Two of the aspartokinases of E. coli are parts of bifimctional enzymes, which also contain the homoserine dehydrogenases that are needed to reduce aspartate semialdehyde in the third step. These aspar-tokinase-homoserine dehydrogenases 1 and 11 (Fig. 

Isoenzymes. In this case, multiple enzymes are made each carries out the same reaction but is regulated by a different end product. This mechanism is used in both feedback inhibition and feedback repression. A well known example of such control is the aspartic acid family in E. coli where the three aspartokinases are regulated by lysine, threonine and methionine respectively (Stadtman, 1968). 

Concerted The first common step is not susceptible to feedback inhibition by excess of only one end product, E or G but is inhibited by simultaneous presence of excess of E and G. Rhodopseud. capsulatus Aspartokinase is inhibited by excess amount of Lys and Thr but not by Lys or Thr tilone. 

Several plant aspartate kinases are activated by other amino acids such as valine, alanine, and isoleucine (Table HI). Activation does not appear to be a general effect of hydrophobic amino acids, since neither methionine nor leucine influence the activity of the maize enzyme yet, leucine has been reported to activate the enzyme isolated from Sinapsis alba and inhibit the enzyme from Helianthus annus. Interaction of these secondary effectors with the various aspartate kinases has not been fully explored, but several observations suggest a considerable degree of complexity. Alanine partially relieves threonine inhibition of the enzyme isolated from pea seedlings (Aames and Rognes, 1974). Lysine inhibition of maize aspartokinase is diminished in the presence of isoleucine, alanine, or valine (Bryan e/ al., 1970) and threonine, even though it does not inhibit the maize enzyme, counteracts... 

The scheme illustrated in Fig. 6 is tentative, and the possibility that a number of other plausible control mechanisms may operate in plants has not been adequately explored. For example, in spite of the established importance of AdoMet in the control of methionine biosynthesis in microorganisms, this compound has frequently not been considered in studies of potential effectors of plant enzymes (e.g., aspartokinase). Another possibility is that methionine biosynthesis may be controlled by changes in the amounts of enzymes (e.g., by induction, repression, etc.) rather than by changes in the activities of enzymes (e.g., by feedback stimulation or inhibition). 

H. (1961) Feed-back inhibition and repression of aspartokinase activity in Escherichia coli and Saccha-romyces cerevisiae. Biol Chem.,... 

In E. coli there are three aspartokinases I (coded by thrA), II (coded by met L), and III (coded by lysC) and two homoserine dehydrogenases I and II that are inhibited or repressed by only one or two amino acids of the aspartate family which ensures that pathway is not shut down with the excess of one product. Each amino acid regulates the first enzyme in its branch to maintain the proper ratio of the amino acids. In Corynebacterium, the regulation is much simpler with only one aspartokinase and here the amino acid biosynthesis is controlled by the synergistic action of the end products. 

Kato C, Kurihara T, Kobashi N, Yamane H, Nishiyama M (2004) Conversion of feedback regulation in aspartate kinase by domain exchange. Biochem Bioph Res Co 316 802-808 Kim YH, Park JS, Cho JY, Cho KM, Park YH, Lee J (2004) Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli. Biochem J 381 823-829 Klaffl S, Eikmanns BJ (2010) Genetic and functional analysis of the soluble oxaloacetate decarboxylase from Corynebacterium glutamicum. J Bacterid 192 2604-2612 Komatsubara S, Kisumi M, Murata K, Chibata 1 (1978) Threonine production by regulatory mutants of Serratia marcescens. Appl Environ Microbiol 35 834-840 Kotaka M, Ren J, Lockyer M, Hawkins AR, Stammers DK (2006) Structures of R- and T-state Escherichia coli aspartokinase 111 mechanisms of the allosteric transition and inhibition by lysine. J Biol Chem 281 31544-31552... 

The intermediate of L-lysine biosynthesis, L-aspartyl-semialdehyde, is also a precursor for the biosynthesis of L-threonine, L-isoleucine, and L-methionine. Homoserine dehydrogenase catalyses NADPH-dependent reduction of L-aspartyl-semialdehyde to L-homoserine. Flux from L-aspartyl-semialdehyde toward L-homoserine could be reduced by introducing alleles for less active homoserine dehydrogenase variants. In addition, L-lysine production increased as the prevailing threonine concentrations in such horn mutants were too low for feedback inhibition of aspartokinase [67, 68]. 

The pathway of biosynthesis of L-lysine and L-threonine including controls of the biosynthesis in Corynebacterium glutamicum is shown in O Fig. 4.8. The formation of phosphoaspartate from aspartate is the first step and is catalyzed by aspartokinase. The activity of this enzyme is controlled through concerted feedback inhibition by L-lysine and L-threonine. 

Fine Regulation by Isoenzymes. We have just mentioned the end product inhibition of threonine deaminase by isoleucine. Isoleucine belongs, together with threonine, methionine, and lysine, to the aspartate family of amino acids (Fig. 118). The synthetic pathway to these amino acids starts with the conversion of aspartate into aspartyl phosphate which is catalyzed by aspartokinase. Later the initially undivided synthetic pathway branches out to lead to the amino acids mentioned (Fig. 157). This branching presents intracellular regulation with a serious problem. For example, more than sufficient threonine might be present in the cells. 

The further supply of threonine could then be easily stopped by end product repression or end product inhibition of aspartokinase. However, the synthesis of methionine and lysine would also be stopped, although both of these amino acids might be urgently needed. The solution to the problem lies in the fact that three isoenzymes of aspartokinase have been found in E. coli, one of which can be blocked by threonine and a second by lysine, by end product repression or inhibition. The activity of the third isoenzyme of aspartokinase is also subject to appropriate regulation somewhat later in the synthetic pathway. Although these findings were made in bacteria there are various indications that the situation in higher plants is likely to be quite similar. 

The reactions catalyzed by aspartokinase (1) and aspartate semialdehyde dehydrogenase (2) are utilized for the synthesis of all pathway products, including threonine. Regulation of aspartokinase activity is, therefore, considered to be of central importance in the overall control of the pathway. Two classes of differentially regulated isozymes of aspartokinase have been isolated from plants. One class is comprised of enzymes subject to feedback inhibition by threonine the other encompasses those inhibited by lysine, or by lysine and 5 -adenosylmethionine. Examples of each class have been isolated from several... 

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