Malate from fumarate

Major groove (DNA), 1104-1105 Malate, from fumarate, 221-222 MALDI-TOF mass spectrometry, 417-418... 

L-Malate (from fumarate) Gibberellic acid (from cheese whey) 2,3-Butanediol (from whey permeate) L-Glutamic add Fumarase Brevibacterium ammonia genes Fusarium moniUform cells Klebsiella pneumoniae cells Brevibacterium flavum (multienzyme)... 

Two of the enzymes of the tricarboxylic acid cycle, aconitase and fumarase, catalyze reactions in which water is added reversibly to an unsaturated polycarboxylic acid. Both enzymes exhibit rigid stereospecificity fumarase forms only L-malate from fumarate and forms only fumarate (trans) and not maleate (czs-ethylenedicarboxylic acid), and aconitase reacts with only cis-, not imns-aconitate, and with D-, not L-isocitrate. Citrate is a symmetrical molecule, with no optical isomers, but it will be shown that steric factors also enter into the reaction of this substrate with aconitase. The enzymes of the tricarboxylic acid cycle, in contrast to the glycolytic enzymes, are associated with intracellular granules known as mitochondria. Studies of the individual enzymes have depended to a large extent on the separation of soluble activities from these particles. Aconitase and fumarase are released from the particles very rapidly under mild conditions often in the preparation of cell-free homogenates these activities are largely solubilized, and special care must be taken to demonstrate their origin in mitochondria. 

Malate From Fumarate 161 Potential for Biodegradable Plastic ... 

This enzyme is highly stereospecific it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate). In the reverse direction (from L-malate to fumarate), fumarase is equally stereospecific D-malate is not a substrate. 

The second substrate glyoxylate approaches from the other side of the molecule and condenses as is shown. Since any one of the three protons in either R or S chiral acetyl-CoA might have been abstracted by base B, several possible combinations of isotopes are possible in the L-malate formed. One of the results of the experiment using chiral (R) acetyl-CoA is illustrated in Eq. 13-43. The reader can easily tabulate the results of removal of the 2H or 3H. However, notice that if the base -B removes 2H (D) or 3H (T) the reaction will be much slower because of the kinetic isotope effects which are expected to be Hk/"k 7 and "k/ k = 16. A second important fact is that the pro-R hydrogen at C-3 in malate is specifically exchanged out into water by the action of fumarate hydratase. From the distribution of tritium in the malate and fumarate formed using the two chiral acetates, the inversion by malate synthase was established. See Kyte231 for a detailed discussion. 

FIG. 21 Schematic diagram for the production of L-malic acid from fumaric acid using a six-compartment (Zi-Z6) ED stack composed of anionic (a) and cationic (c) membranes, as extracted from Sridhar (1987). As ammonium fumarate is formed, it is enzymatically converted into ammonium malate in an external bioreactor (R). The combined system is also provided with a series of storage tanks for the acidic cathode-(Dl) and anode-(D6) rinsing solutions, raw materials (D2), ammonium fumarate (D3), ammonium malate (D4), and final product (D5). 

Further proof that COj is assimilated by means of the enzyme oxalacetate /3-carboxylase was obtained by Evans and coworkers, who succeeded in preparing a cell-free preparation of this enzyme from liver. The enz3rme was able to catalyze the decarboxylation of oxalacetate to pyruvate. These investigators were able to demonstrate an uptake of C Oj. Utter and Wood have demonstrated conclusively, however, that in the presence of isotopic CO2, pyruvate can be converted to oxalacetate containing isotopic carbon and that the process is, of course, reversible. Addition of adenosine triphosphate to this liver enzyme system increased the rate of incorporation of C Os. Wood, Vennesland and Evans S have also shown that during the fixation of C02, isotopic carbon is incorporated solely and in equal concentrations into the carboxyl groups of pyruvate, lactate, malate and fumarate. 

Wood and coworkers have demonstrated that pigeon liver mince poisoned with malonate is unable to convert C 02 into isotopic succinate but can direct the isotope into malate and fumarate as well as into the a-carboxyl of a-ketoglutarate (see Fig. 5). On the other hand, non-poisoned liver possesses the ability to anaerobically fix C 02 in succinic acid as well as fumaric and malic acids. The dicarboxylic acids invariably contain the isotopic carbon predominantly in their respective carboxyl groups. These important experiments provide conclusive proof for two main concepts. First, that malonate specifically inhibits succinic acid dehydrogenase and hence blocks completely the formation of succinic from fumaric acid. Secondly, there must be two routes by which succinate is formed. By the first pathway, pyruvate can condense with... 

Fumarase is an enzyme component of the TCA cycle that catalyzes the reversible reaction of fumarate to L-malate with equilibrium favoring malate production. It is a soluble enzyme with high turnover number. In one report, fumarate content in some organisms can be as high as lOOOmg/kg of wet cells [80]. Theoretically, a malate weight yield of 115% can be obtained from fumarate. However, in reality, a weight yield of 90-95% is often obtained. 

The stereospecific formation of the S enantiomer of malic acid from fumaric acid also occurs in the tricarboxylic acid (TCA) cycle. The (5)-(-)-malic acid that is produced is converted to oxaloacetic acid by the enzyme malate dehydrogenase. 

In the reductive pathway, the Krebs cycle enzymes are assumed to operate as far as a-oxoglutarate, thus forming a linear pathway. A second linear pathway, from oxaloacetate to malate to fumarate to succinate, is suggested to account for the formation of succinic acid [46]. In support of this new pathway are the observations that (/) yeast contains cytoplasmic malate dehydrogenases capable of converting oxaloacetate to malate, (//) several fumarate reductases (FAD-dependent) have been found in the yeast cytoplasm which have high affinity for fumarate and are unable to oxidize succinate [52] and (Hi) succinate is a significant product of fermentation, i.e. an end product . 

Fig. 17.11 Summary of carbohydrate metabolism in yeast growing anaerobically, i.e. in fermentation. The dotted lines represent the oxidative pathway for the formation of succinate (and possibly fumarate and malate) from pyruvate. The alternative reductive pathway is indicated by solid lines. 

The reaction of CO2 fixation onto phosphoenolpyruvic acid by PEP carboxytransphosphorylase is considered (O Brien and Wood, 1974) as a control mechanism of propionic acid fermentation. They observed a conversion of the enzymatically active tetrameric form of PEP carboxytransphosphorylase isolated from P. shermanii into a less active dimeric form induced by oxalate, malate and fumarate. Therefore, the loss of activity by enzyme dissociation, accompanied by increased proteolysis, is an effective means of controlling the level of intermediates in propionic acid fermentation. Differential abilities of propionibacteria to fix CO2 could be associated (Wood and Leaver, 1953) with their abilities to carry out the reaction C02 Ci and to form sulfhydryl complexes with Ci. 

Figure 17.15 Major metabolic pathways involved in SA production in Saccbaromyces cerevisiae. Bold arrows indicate the major routes for succinate synthesis starting from glucose (a) via the reductive TCA cycle and (b) via the giyoxyiate cycle. PEP, phos-phoenolpyruvate OAA, oxaloacetate MAL, malate FUM, fumarate Suc-CoA, sucdnyl-CoA cr-KG, cr-ketoglutarate ICT, isodtrate CIT, citrate, ppc, PEP carboxykinase pyc, pyruvate carboxylase pyk, pyruvate kinase ...

The substrate, L-aspartate, is produced from fumarate by an enzyme system involving aspartase, as described in the section on L-aspartate. To produce L-alanine directly from fumarate, the L-alanine-producing column was connected in tandem to an L-aspartate-producing column. In this tandem column system, side reactions caused by fumarase in Escherichia coli and alanine racemase in P. dacunhae reduced the yield. Then, both bacterial cells were separately treated with high temperature and low pH, respectively, and the enzymes responsible for the side reactions were inactivated. Immobilization of these two kinds of bacterial cells with K-carrageenan resulted in the production of L-alanine in a single reactor without the production of the side products, malate and o-alanine (Takamatsu et al. 1982 Chibata et al. 1986b). 

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