Threonine dehydratase reactions

FIGURE 6-28 Feedback inhibition. The conversion of L-threonine to L-isoleucine is catalyzed by a sequence of five enzymes (E, to E5). Threonine dehydratase (E,) is specifically inhibited allosterically by L-isoleucine, the end product of the sequence, but not by any of the four intermediates (A to D). Feedback inhibition is indicated by the dashed feedback line and the symbol at the threonine dehydratase reaction arrow, a device used throughout this book. 

One of the most distinguishing features of metabolic networks is that the flux through a biochemical reaction is controlled and regulated by a number of effectors other than its substrates and products. For example, as already discovered in the mid-1950s, the first enzyme in the pathway of isoleucine biosynthesis (threonine dehydratase) in E. coli is strongly inhibited by its end product, despite isoleucine having little structural resemblance to the substrate or product of the reaction [140,166,167]. Since then, a vast number of related... 

L-Serine dehydratase [EC 4.2.1.13], also known as serine deaminase and L-hydroxyaminoacid dehydratase, catalyzes the pyridoxal-phosphate-dependent hydrolysis of L-serine to produce pyruvate, ammonia, and water. In a number of organisms, this reaction is also catalyzed by threonine dehydratase. 

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps (Fig. 6-28). In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by isoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine. 

Pyridoxal Phosphate Reaction Mechanisms Threonine can be broken down by the enzyme threonine dehydratase, which catalyzes the conversion of threonine to a-ketobutyrate and ammonia. The enzyme uses PLP as a cofactor. Suggest a mechanism for this reaction, based on the mechanisms in Figure 18-6. Note that this reaction includes an elimination at the j8 carbon of threonine. 

This reaction is readily reversible. Another means of metabolizing serine, which accounts for its glucogenic character, as well as that of glycine, is the conversion of serine to pyruvate, as indicated in Figure 20.12. This reaction is catalyzed by serine dehydratase. A similar enzyme, threonine dehydratase, converts threonine to a-ketobutyrate, and the latter is then converted to propionyl-CoA, as indicated in Figure 20.13. Another similar enzyme, cysteine desulfhydrase, con-... 

A similar stereochemical question as in the /8-replacement reactions can be asked in the a, /8-eliminations where the group X is replaced by a hydrogen, i.e., is the proton added at C-/8 of the PLP-aminoacrylate on the same face from which X departed or on the opposite face This question has been answered for a number of enzymes which generate either a-ketobutyrate or pyruvate as the keto acid product. Crout and coworkers [119,120] determined the steric course of proton addition in the a,/8-elimination of L-threonine by biosynthetic L-threonine dehydratase and of D-threonine by an inducible D-threonine dehydratase, both in Serratia marcescens. Either substrate, deuterated at C-3, was converted in vivo into isoleucine, which was compared by proton NMR to a sample prepared from (3S)-2-amino[3-2H]butyric acid. With both enzymes the hydroxyl group at C-3 was replaced by a proton in a retention mode. Although this has not been established with certainty, it is likely that both enzymes, like other bacterial threonine dehydratases [121], contain PLP as cofactor. Sheep liver L-threonine dehydratase, on the other hand, is not a PLP enzyme but contains an a-ketobutyrate moiety at the active site [122], It replaces the hydroxyl group of L-threonine with H in a retention mode, but that of L-allothreonine in an inversion mode [123]. Snell and coworkers [124] established that the replacement of OH by H in the a, /8-elimination of D-threonine catalyzed by the PLP-containing D-serine dehydratase from E. coli also proceeds in a retention mode. They... 

Trapping of the aminoacrylate intermediate in the reactions catalyzed by cystathionine-y-synthase and y-cystathionase produced the same diastereomer of KEDB which was different from the one formed with bacterial L-threonine dehydratase. Unfortunately, this experiment has apparently not been done with threonine synthetase. 

Threonine dehydratase catalyzes the formation of 2-ketobutyrate from threonine, and pyruvate from serine. This assay can be used to examine the reaction in the presence of both substrates. 

Serine and threonine dehydrases. Serine and threonine are not substrates in transamination reactions. Their amino groups are removed by the pyridoxal phosphate-requiring hepatic enzymes serine dehydratase and threonine dehydratase. The carbon skeleton products of these reactions are pyruvate and a-keto-butyrate, respectively. 

Valine, leucine, and isoleucine - The synthetic pathway from threonine and pyruvate to valine, leucine and isoleucine is outlined in Figure 21.26. The last four reactions in the biosynthesis of valine and isoleucine are catalyzed by the same four enzymes. Threonine dehydratase, which catalyzes the first step in conversion of threonine to isoleucine, is inhibited by isoleucine. Leucine, isoleucine, and valine are all catabolized via transamination followed by oxidative decarboxylation of the respective keto-acids (see here) and oxidation. The oxidation is similar to fatty acid oxidation, except for a debranching reaction for each intermediate. 

Although the tryptophan synthetase and tryptophanase reactions have been the best studied replacement and 0 eUmination-deamination reactions, others pf special interest are D-serine dehydratase [75-77] from E. coli, D-threonine dehydratase and L-threonine dehydratase from Serratia marcescens [78]. The only information available on the above enzymes is that in these cases also, the events at occur with retention of configuration. 

Fig. 38.5. Summary of the sources of NH4 for the urea cycle. All of the reactions are irreversible except glutamate dehydrogenase (GDH). Only the dehydratase reactions, which produce NH4 from serine and threonine, require pyridoxal phosphate as a cofactor. The reactions that are not shown occurring in the muscle or the gut can all occur in the liver, where the NH4 generated can be converted to urea. The purine nucleotide cycle of the brain and muscle is further described in Chapter 41.

The presence of two genes, nisB and nisC, encoding 993- and 414-residue proteins without significant homology to other known proteins, but conserved in several lantibiotic operons, has made them strong candidates for post-trans-lational modifications in the maturation pathway of lantibiotics [40]. Limited similarity between NisB and E. coli IlvA, a threonine dehydratase, was reported and hence a dehydratase function for NisB was suggested [40]. Mutation studies of NisB, NisC, EpiB, EpiC, and SpaB indicated that these proteins were essential for nisin, epidermin and subtilin biosynthesis, respectively [40,86,87,190]. As no precursors have been identified and characterized in these mutants, conclusions about the reaction that is catalyzed by these proteins remain speculative [40]. Secondary-structure predictions and experimental evidence confirmed that NisB and SpaB are both membrane-bound [100]. 

Fig. 6. Effects of substrate, isoleucine, valine, and pH on the activity of maize threonine dehydratase. The enzyme was isolated from shoots of etiolated seedlings and partially purified by ammonium sulfate fractionation. Activity was measured as described by Datta (1971) under conditions in which velocity was proportional to the amount of enzyme added and linear over the time of measurement (10 min). The assay mixtures contained EPPS (0.1 M) as buffer and 0.2 M KCI. The pH of complete reactions mixtures was determined with an Orion Model 70IA pH meter and did not change during the incubations at 30°C. Further information will be published elsewhere by E. Lissik and J. Bryan. Solid circles represent control measurements. L-Valine (1.0 mM) was added to the assays indicated by open symbols and dashed lines. Solid and open triangles represent measurements in the presence of L-isoleucine. Isoleucine concentration was 10 txM for the assays at pH 7.45 and 20 juAf during measurements at pH 7.95 and 8.70.

In bacteria and mammals reaction 1 is readily catalyzed by the enzyme serine dehydratase. Sharma and Mazumder (1970) have purified an enzyme from spinach leaves which will act as both a serine dehydratase or a threonine dehydratase (E.C. 4.2.1.16). The latter appears to be the preferred substrate, but the presence of such an enzyme would maire possible the direct conversion of serine to pyruvate which could then enter the TCA cycle. 

The enzyme catalyzing reaction 1 is threonine dehydratase. As described above for serine dehydratase, an enzyme having activity towards both serine and threonine has been found in spinach leaves (Sharma and Mazumder, 1970). Dougall (1970) has also reported a threonine dehydratase in extracts from Paul s Scarlet Rose tissue cultures. The oxidative decarboxylation of the 2-oxobutyrate would lead to propionate (reaction 2) which could then be oxidized via the pathway demonstrated by Giovanelli and Stumpf (1958). 

Davies L. Functional and stereochemical specificity at the carbon atom of substrates in threonine dehydratase-catalyzed a.P elimination reactions. J Biol Chem 1979 254 4126-4131. 

The initial reaction associated with isoleucine biosynthesis is catalyzed by threonine dehydratase (18). In microorganisms and a few plants, both degra-dative and biosynthetic enzymes have been identified. The former are not regulated by amino acids, but some are activated by AMP or ADP. An apparently unregulated threonine dehydratase has been purified from seeds of Cus-cuta campestris (Madan and Nath, 1983). The lack of identifiable regulatory phenomena precludes identification of the function of this enzyme as either biosynthetic or degradative in nature. 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

In addition to glutamate, a number of amino acids release their nitrogen as NH4 (see Fig. 38.5). Histidine may be directly deaminated to form NH4 and urocanate. The deaminations of serine and threonine are dehydration reactions that require pyridoxal phosphate and are catalyzed by serine dehydratase. Serine forms pyruvate, and threonine forms a-ketobutyrate. In both cases, NH4 is released. 

Some amino acids, such as serine and threonine, possess a hydroxyl group on their fi carbon. They can be directly deaminated by dehydration. A dehydratase catalyses this reaction, producing the corresponding ketonic acid and ammonium (Figure 2.27). 

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