L-Threonine dehydratase

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

The first enzyme in the sequence, L-threonine dehydratase, is strongly inhibited by L-isoleucine, the end product, but not by any other intermediates in the sequence. 

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. 

Serine Racemase (EC 5.1.1.16] Serine racemases have been discovered in both bacteria and eukaryotes (for a review see [60, 62). In the latter organisms, serine racemase catalyzing the conversion of L-Ser to D-Ser was at first discovered in the silkworm Bombyx mori it is a PLP-dependent racemase which is also active on L-Ala (-6% of the activity on L-Ser). A serine racemase was also purified from rat brain (and a serine racemase cDNA was cloned from mouse brain). Mammalian serine racemase shows sequence simUarily with L-threonine dehydratase from various sources all the active site residues of the latter enzyme are also conserved in mouse serine racemase. Mammalian serine racemase is a member of the fold-type II group of PLP enzymes (similarly to L-threonine dehydratase, D-serine dehydratase, and so on) and distinct from alanine racemase, which belongs to the fold-type III group. Mouse serine racemase shows a low kinetic efficiency the Km values for L- and D-Ser are -10 and 60 mM, respectively and the V ax values with L- and D-Ser are 0.08 and 0.37 units/mg protein (less than 0.1% of those of alanine racemase on L- and D-Ala, see above). 

L-Serine and L-threonine dehydratases dehydrate and subsequently deaminate the amino add to the corresponding a-keto add. These enzymes are known to require pjn-idoxal-S -phosphate as a coenzyme. They can function in a biosynthetic or catabolic marmer (99). Both enzymes can cause problems for the whole-cell-based production of L-serine (100). 

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. 

Desai, I.D., Laub, D. and Antia, N.J., 1972. Comparative characterization of L-threonine dehydratase in seven species of unicellular marine algae. Phytochemistry, 11 277— 287. 

In a study of differential labeling of the allosteric site for 5 -ADP binding in the L-threonine dehydratase of Clostridium tetanomorphum, six reagents were compared for their ability to produce a loss in ADP binding in the absence or in the presence of ADP as a protective ligand. 

Thiouridine monophosphate, 89 L-Threonine dehydratase, 64 D-Threose 2,4-bisphosphate, 21 Thrombin, 128, 206, 207 Thymidine kinase, 89 Thymidylate synthetase, 164, 307-312 L-Thyroxine, analog, 435 Thydroxyl-JV-carbonylmethylprealbumin, 436... 

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. 

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. 

A classic example of allosteric inhibition is the case of the enzymatic conversion of L-threonine into L-isoleucine by bacteria. The first of five en mes, threonine dehydratase is inhibited by the end product, isoleucine. This inhibition is very specific, and is accomplished only by isoleucine, which binds to a site on the enzyme molecule called the regulatory, or allosteric, site. This site is different from the active site of the en2yme, which is the site of the catalytic action of the eri2yme on the substrate, or molecule being acted on by the eri2yme. 

Both the l- and D-serine deaminase catalyze the elimination of the amino functionality of both l- and D-serine, but the mechanism proceeds via the initial elimination of water and these enzymes are thus classified as hydrolyases (l- and D-serine dehydratases E.C. 4.2.1.13 and E.C. 4.2.1.14, respectively)[27, 28. The aminoacrylate generated is unstable and subsequent elimination of the amine results in the formation of pyruvate. Similarly, threonine deaminase is in effect a dehydratase that converts L-threonine into 2-oxobuturate, water and ammonia (E.C. 4.2.1.16) (Scheme 12.6-1). 

L-Threonine is an absolute dietary essential amino acid. The degradation of the threonine can be via a dehydratase or a specific aldolase in the liver. 

The enzyme threonine dehydratase (EC 4.2.1.16) has been shown to dehydrate both L-threonine 128a and L-allothreonine 129 in to yield (3R)-[3- H,]-a-ketobutyrate 124 (131) (Scheme 40), the configuration of which was proven by conversion to (2R)-[2- H,]propionate and comparison of the ORD with that of an authentic sample. This implies either that the bound substrates 128a and 129 dehydrate with different stereochemistries and protonate from the same side or that they dehydrate in identical fashion and protonate from different sides. Threonine dehydratase has an important role in the biosynthesis of valine, as we shall see in Section VIII. 

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.

L-lsoieucine, lie L-a-amino-P-methylvaleric acid, CH3-CH2-CH(CH3)-CH(NH2)-C00H, an aliphatic, neutral amino acid found in proteins. He is found in relatively large amounts in hemoglobin, edestin, casein and serum proteins, and in sugar beet molasses, from which it was first isolated in 1904 by F. Ehrlich. It is an essential dietary amino acid, and is both glu-coplastic (degradation via propionic acid) and keto-plastic (formation of acetate) (see Leucine), The biosynthesis of He starts with oxobutyrate and pyruvate. Oxobutyrate is synthesized by deamination of L-threonine by threonine dehydratase (threonine de-... 

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. 

Faleev NG, Martinkova NS, Sadovntkova MS, Sapoiovskaya MB, Belikov VM. The L- and p-threonine dehydratases accompanying L-tyiosine phenol-lyase Selective decomposition of D-threonine in racemic mixture. Enz Microb Technol 1982 4 164-168. 

Croui DHG, Gregorio MVM, Muller US. Komatsubata S, Kisumi M. Chibata O. Stereochemistry of the conversions of L-threonine and P-threonine into 2-OXobutanoate by the L-threonine and D-chreonine dehydratases of Serratia marcescens. Eur Biochem 1980 106 97-105. 

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