Threonine deaminase, inhibition

Fig. 3. Generation of propionyl-CoA from the isoleucine biosynthetic pathway. The intermediate 2-ketobutyrate can be decarboxylated by either the 2-oxoacid dehydrogenase complex or at low efficiency by the pyruvate dehydrogenase complex. Inhibition of the threonine deaminase by isoleucine and of the acetolactate synthase by herbicides are indicated with dashed arrows...

It was snbseqnently discovered that the first enzyme in the pathway for isoleucine synthesis, which is threonine deaminase, was inhibited by isoleucine in an extract of E. coli. No other amino acid caused inhibition of the enzyme. Threonine deaminase is, in fact, the rate-limiting enzyme in the pathway for isoleucine synthesis, so that this was interpreted as a feedback control mechanism (Fignre 3.13(a)). Similarly it was shown that the hrst enzyme in the pathway for cytidine triphosphate synthesis, which is aspartate transcarbamoylase, was inhibited by cytidine triphosphate (Fignre 3.13(b)). Since the chemical structures of isoleucine and threonine, or cytidine triphosphate and aspartate, are completely different, the qnestion arose, how does isolencine or cytidine triphosphate inhibit its respective enzyme The answer was provided in 1963, by Monod, Changenx Jacob. 

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. 

Consider, for example, the biosynthesis of the amino acids valine, leucine, and isoleucine. A common intermediate, hydroxy ethyl thiamine pyrophosphate (hydroxy ethyl-TPP Section 17.1.1). initiates the pathways leading to all three of these amino acids. Hydroxyethyl-TPP can react with a-ketobutyrate in the initial step for the synthesis of isoleucine. Alternatively, hydroxyethyl-TPP can react 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 deaminase, the PLP enzyme that catalyzes the formation of a-ketobutyrate, is allosterically inhibited by isoleucine (Figure 24.22). This enzyme is also allosterically activated by valine. Thus, this enzyme is inhibited by the product of the pathway that it initiates and is activated by the end product of a competitive pathway. This mechanism balances the amounts of different amino acids that are synthesized. 

Figure 24.22. Regulation of Threonine Deaminase. Threonine is converted into a-ketobutyrate in the committed step leading to the synthesis of isoleucine. The enzyme that catalyzes this step, threonine deaminase, is inhibited by isoleucine and activated by valine, the product of a parallel pathway.

Endproduct inhibition Inhibition of threonine deaminase by isoleuclne E. coli) (1)... 

Inhibitor of the Initial enzyme of the pathway. The Inhibition of threonine deaminase Is a well known example to which I refer later In this article (1 ). There are variations on this general theme in which multiple endproducts may act In concert or syner-glstlcally upon a single enzyme catalyzing the first step in a common pathway or In which a segment of a single pathway is controlled by an intermediate Inhibiting an earlier step [e.g., the inhibition of phosphofructoklnase of Escherichia coli by phospho-enolpyruvate ( )]. 

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. 

It is perhaps interesting to note that two entirely different types of thiaisoleucine-resistant mutants were obtained from E. coli and S. typhimurium. All the thiaisoleucine-resistant mutants of the latter were found to have threonine deaminases that were less sensitive to isoleucine inhibition than was the wild-type enzyme. These organisms have not been studied further except to verify that the lesion, as expected, is closely linked to the threonine deaminase structural gene ilv A) (J. M. Blatt, unpublished observations, 1970). In E. coli, however, all the thiaisoleucine-resistant mutants examined had derepressed... 

Umbarger and Brown (161) established that valine and isoleucine biosynthesis is controlled by a negative feedback mechanism. In the case of isoleucine this was shown to result from the inhibition of L-threonine deaminase by L-isoleucine. 

An example is the inhibition of threonine deaminase by L-isoleucine. L-Isoleucine is the end product of a synthetic pathway, the first enzyme of which is threonine deaminase (Fig. 156). In end product inhibition the steric configuration of a protein, i.e. an enzyme protein, is altered. The steric configuration of a protein is also changed in end product repression. 

Fig. 156. End product inhibition inhibition of threonine deaminase by isoleucine.

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. 

Competitive inhibitors are inhibitors which have an effect on the but not on the V of an enzyme-catalysed reaction. The V is unchanged because the number of functional active sites is not altered but a greater substrate concentration is required to achieve the maximum utilization of the sites. Consequently, the for the substrate increases. Competitive inhibition may be overcome by the addition of more substrate to the enzyme reaction mixture. Competitive inhibitors often bear a structural similarity to the substrate and compete with the substrate for the active sites of the enzyme, i.e. they are isosteric. However, competitive inhibitors are not necessarily structurally analogous to the substrate, e.g. salicylate inhibition of 3-phospho-glycerate kinase, and may bind to a site distinct from the active site, e.g. L-isoleucine inhibition of threonine deaminase from Escherichia coli. The classical example of competitive inhibition is the action of malonate on succinate dehydrogenase (Figure 6.9) which advanced the elucidation of the... 

By this means, it has been found that the excess of L-isoleucine has two distinct effects—one that is relatively slow, and unothcr that is rapid. The slower effect is to repress production by the cell of all the enzymes required io catalyze the series of biochemical reactions in the metabolic pathway by which the cell synthesizes L-isoleucine. The Iasi effect is to inhibit production of the enzyme for the first reaction ill the series. This enzyme is L-thrconinc deaminase, which removes the amino group from L-threonine. as a preliminary step to iis oxidation and reimroduction of (he amino group, in order to produce L-isolcucine from it. 

Umbarger and Brown 233) have observed L- and D-threonine dehy-drases in E. coli which are stimulated by pyridoxal phosphate. In a continuation of this work 234) they have presented evidence that E. coli extracts contain two distinct L-threonine dehydrases. One of the dehydrases is the L-threonine dehydrase of Wood and Gunsalus and requires pyridoxal phosphate, AMP, and glutathione. It was found to be an adaptive enzyme, and was active against L-serine. The second dehydrase is present in extracts of wild-type E. coli, but not in mutants which are unable to convert threonine to a-ketobutyrate as a step in isoleucine synthe. It requires only pyridoxal-5-phosphate and is inhibited by isoleucine. On the basis of kinetic studies it is proposed that the Wood-Gunsalus dehydrase combines with one molecule of substrate, whereas the other enzyme combines with two. L-Serine is a substrate for both enz3unes. Evidence is presented for a third deaminase in E. coli cells which is serine-specific. 

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