Aspartate kinase inhibition

The L-threonine biosynthetic pathway consists of five enzymatic steps from L-aspartate. E. coli has three aspartate kinase isoenzymes, key enzymes which catalyze the first reaction of the L-threonine biosynthetic pathway. The aspartate kinase isoenzymes I, II, and III encoded by the thrA, metL, and lysC genes, respectively, are affected by feedback inhibition by L-threonine, L-methionine, and L-lysine, respectively. C. glutamicum has only one aspartate kinase encoded by the lysC gene, which is subjected to feedback inhibition by L-lysine and... 

It was shown that this kinase domain does not exhibit basal activity and that it is autoinhibited in a dual way [17]. On the one hand, the ATP binding site is blocked by the C-terminal regulatory tail (shown in red in Fig. 15.1), preventing binding of the cosubstrate ATP. On the other hand, the cataMic aspartate is inhibited by tyrosine-170 (TYR170) so that phosphorylation of... 

Ramos, C. Delgado, M.A. Calderon, I.L. Inhibition by different amino acids of the aspartate kinase and the homoserine kinase of the yeast Sac-charomyces cerevisiae. FEBS Lett., 278, 123-126 (1991)... 

Fig. 10. Schematic representation of the split biosynthetic pathway of L-lysine in wildtype Corynebacterium glutamicum including the branch point of aspartate semialdehyde distribution. The metabolites derived from the aldehyde via the synthase activity are D,L-di-aminopimelate and L-lysine, whereas that resulting from dehydrogenase activity are L-threo-nine, L-methionine, and L-isoleucine. The activity of the dehydrogenase is inhibited at elevated L-threonine concentrations and its synthesis is repressed by L-methionine. Accumulating intracellular lysine causes feedback inhibition of aspartate kinase and activates lysE transcription...

Problems associated with identification of specific regulatory mechanisms can be illustrated by a brief consideration of changes in the apparent levels of aspartate kinase during plant growth. Aspartate kinase extracted from whole carrot roots, freshly sliced root tissue, or recently subcultured cell suspensions is predominantly sensitive to inhibition by the pathway product, threonine. However, when root slices are incubated in a growth stimulating medium or when suspension cultures are allowed to grow for a period of time, the bulk of the extractable aspartate kinase activity is sensitive to... 

Inhibitor constants (A j) or the concentration of inhibitor required for half-maximal inhibition under specified assay conditions ffo.s) provide an indication of the quantitative sensitivity of an enzyme. Such indicators of plant aspartate kinase sensitivity are consistently below 1.0 mM and in several instances are less than 100 fiM (Table III). Even though the effective concentration of regulatory metabolites in plant cells is difficult to estimate (Section III,C), the demonstrable inhibitory effects of comparatively low concentrations of lysine or threonine on aspartate kinase i/t vitro suggest that enzyme activity is likely to be regulated by these pathway products in vivo. 

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... 

Fig. 7. Sequential control of the synthesis of the aspartate family of amino adds. Temporal control of the flow of carbon is illustrated by the successive Figs. 1-5. Potential quantitative changes in flow are approximated by the thickness of the solid arrows. As the concentration of an end product is increased (indicated by closed boxes), the pattern of synthesis is altered by utilization of negative (-) or positive (+) regulatory mechanisms as described in the text. The pattern of control which is illustrated assumes that aspartate kinase is sensitive to inhibition only by lysine. Variations of this pattern are discussed in the text.

Aspartate kinase is usually subject to very strong inhibition by lysine and/or threonine (Miflin et ai, 1979). Even if the worker can be certain that the activity that is measured is due to the action of asparagine synthetase, there is still no information on whether glutamine or ammonia is acting as the amino donor. It is for these reasons that the hydroxamate assay is not recommended. 

Shiio, I., Miyajima, R., and Sano, K. (1970) Genetically desensitized aspartate kinase to the concerted feedback inhibition in Brevibacterium flavum. 

Lysine plus threonine severely inhibits growth of maize in a synergistic manner. Growth inhibition could result from combined effects of lysine on aspartate kinase and threonine on homoserine dehydrogenase, resulting in starvation for methionine. Growth inhibition by lysine -i- threonine can be overcome by supplying methionine. Bryan [1980) Miflin [1977). 

E. coli aspartate kinase III is subjected to feed-back inhibition by L-lysine. The inhibition is complete (Chassagnole et al. 2001), and the inhibitory mechanism is... 

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... 

Sauer U, Eikmanns BJ (2005) The PEP-pyruvate-oxaloacetate node as the switch point forctubon flux distribution in bacteria. EEMS Microbiol Rev 29 765—794 Schrumpf B, Schwarzer A, Kalinowski J, Puhler A, Eggeling L, Sahm H (1991) A functionally split pathway for lysine synthesis in Corynebacterium glutamicium. J Bacteriol 173 4510-4516 Shiio I, Miyajima R (1969) Concerted inhibition and its reversal by end products of aspartate kinase in Brevibacterium flavum. J Biochem 64 849-859 Shinfuku Y, Sorpitipom N, Sono M, Furusawa C, Hirasawa T, Shimizu H (2009) Development and experimental verification of a genome-scale metaboUc model for Corynebacterium glutamicum. Microb Cell Fact 8 43-57... 

Yoshida A, Tomita T, Kurihara T, Fushinobu S, Kuzuyama T, Nishiyama M (2007) Structural insight into concerted inhibition of a2 32-type aspartate kinase from Corynebacterium glutamicum. J Mol Biol 368 521-536... 

In the case of Brevibacterium flavum, only aspartate kinase is sensitive to the feedback inhibition. 

Regulatory mutants, whose aspartate kinase is desensitized to the concerted feedback inhibition, have been obtained by isolation of lysine analogue-resistant mutants, whose growth was not inhibited by a lysine analogue, such as amino ethyl cysteine (AEC). The lysine analogue behaved as a false feedback inhibitor on aspartate kinase and did not allow growth of the parent strain. Only the mutants desensitized to the feedback inhibition grew. 

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

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