Fumarase malic acid

Optically inactive starting materials can give optically active products only if they are treated with an optically active reagent or if the reaction is catalyzed by an optically active substance The best examples are found m biochemical processes Most bio chemical reactions are catalyzed by enzymes Enzymes are chiral and enantiomerically homogeneous they provide an asymmetric environment m which chemical reaction can take place Ordinarily enzyme catalyzed reactions occur with such a high level of stereo selectivity that one enantiomer of a substance is formed exclusively even when the sub strate is achiral The enzyme fumarase for example catalyzes hydration of the double bond of fumaric acid to malic acid m apples and other fruits Only the S enantiomer of malic acid is formed m this reaction... 

The reaction is reversible and its stereochemical requirements are so pronounced that neither the cis isomer of fumaric acid (maleic acid) nor the R enantiomer of malic acid can serve as a substrate for the fumarase catalyzed hydration-dehydration equilibrium... 

Biosynthesis ofS(— )-M llc Acid. Aqueous fumaric acid is converted to levorotatory malic acid by the intracellular enzyme, fumarase, which is produced by various microorganisms. A Japanese process for continuous commercial production of S(—)-mahc acid from fumaric acid is based on the use of immobilized Brevibacteriumflavum cells in carrageenan (32). The yield of pyrogen-free S(—)-mahc acid that is suitable for pharmaceutical use is ca 70% of the theoretical. 

S. Except for oxido-reductases, transferases, and hydrolases, most ligases (enzymes that catalyze bond formation) are entirely substrate specific. Thus, fumarate hydratase (or fumarase) reversibly and stereospecifically adds water to fumaric acid to produce (S)-( — )-malic acid only (8) (Figure 1), and another enzyme, mesaconase, adds water to mesaconic acid to form (+ )-citramalic acid (9) (Figure 2). Although no extensive studies are available, it appears that neither fumarase nor mesaconase will add water stereospecifically to any other a,(3-unsaturated acid. 

The enzyme fumarase catalyses the stereospecific iram -addition of water to fumaric acid giving (5)-malic acid, and the reverse reaction, the rrans-elimination of water from (S )-malic acid ... 

The addition of water to fiimaric acid catalysed by fumarase is a highly stereospecific reaction and malic acid is formed as the sole product (Figure 2.22, X=H). The ammonia lyase 3-methylaspartase catalyses the similar addition of ammonia to yield L-aspartic acid. When uimatural substrates are used in these reactions (X =/= H), less success is experienced. An increasing X-group gives slow reaction rates. 

L-malic acid by addition of water to fumaric acid by fumarase from Brevibacterium... 

Fumarate hydratase (fumarase), which is discussed in Chapter 13, catalyzes the reversible hydration of fumaric acid to malic acid (Eq. 13-11). It was one of the first enzymes whose pH dependence was studied intensively. A bell-shaped pH dependence... 

Aqueous lumaric acid is converted to levoroialory malic acid by the intracellular enzyme, fumarase. which is produced by various microorganisms. 

The reaction is remarkable for a number of reasons. It is readily reversible and is catalyzed by an enzyme (fumarase) at nearly neutral conditions (pH s 7). Without the enzyme, no hydration occurs under these conditions. Also, the enzymatic hydration is a completely stereospecific antarafacial addition and creates L-malic acid. The enzyme operates on fumaric acid in such a way that the proton adds on one side and the hydroxyl group adds on the other side of the double bond of fumaric acid. This is shown schematically in Figure 10-9. 

Exercise 10-12 The hydration of fumaric acid catalyzed by fumarase in D20 leads to malic acid with only one C-D bond, which is selectively removed when malic acid is enzymatically reconverted to fumaric acid. The configuration of deuteriomalic acid prepared in this way has been shown to correspond to the following projection formula ... 

To diagnose the situation, the malates formed were treated with fumarase. The one formed from (R)-acetyl-CoA became equilibrated with tritiated fumaric acid and retained most of its tritium whereas that from the (S)-acetyl-CoA lost most of its tritium in the reversible dehydration yielding unlabeled fumaric acid (and, then, unlabeled malic acid). Reference to Fig. 58 shows that the former malate was thus 3S and the latter 3R, i.e. the condensation proceeds with inversion of configuration. 

L-Malic acid (HOOC CH2 CHOH COOH) for use in the pharmaceutical industry is manufactured by conversion of fumaric acid by the intracellular enzyme fumarase produced by various microorganisms. The excess fumaric acid is easily separated by crystallization after concentration of the mother solution. Further addition of lime allows malic acid to be separated as calcium malate within a bioreactor crystallizer system. By adding diluted sulfuric or oxalic acid, the salt is split into free malic acid and calcium sulfate or oxalate, the latter being removed by filtration (Mourgues et al., 1997). 

Dicarboxyaziridine (4.c) is a potent competitive inhibitor (Ki = 80 nM) of fumarase, an enzyme that catalyzes the hydration of fumaric acid (4.a) to (S)-malic acid (4.b). Rationalize how 4.c might act as a competitive inhibitor of fumarase. Would you expect the enantiomer of the inhibitor to have a higher or lower K- value Explain. [Greenhut, J., Umezawa, H. Rudolph, F. B. Inhibition of Fumarase by 5-2,3-Dicarboxyaziridine../. Biol. Chem. 1985, 260, 6684-6686.]... 

By application of the CIP rules the order of priority of the atoms directly attached to the chirality centre is O > C(0,0,(0)) > C(C,H,H) > H. The atom or group of lowest priority, hydrogen in this case, is already oriented away from the observer. Therefore the sequence of the remaining three groups can be directly deduced from the formula, and these are easily seen to be arranged in a counter-clockwise sense to the observer. It therefore follows that the formula represents (S)-2-hydroxysuccinic acid (formerly known as L-malic acid). The compound is produced in the citric acid cycle from fumaric acid by fumarate-hydratase (fumarase). 

Fumarase. The development and use of this immobilized enzyme by Tanabe Seiyaku for production of L-malic acid is very similar to that of aspartase ( 3). Lysed Brevibacterium ammoniagenes or B. flavin cells are treated with bile acid to destroy enzymatic activity which converts fumarate to succinate. As with aspartase, the cells can be immobilized in polyacrylamide or k-carrageenan gels. Using a substrate stream of 1 M sodium fumarate at pH 7.0 and 37°C, L-malic acid of high purity has been produced since 1974 by a continuous, automated process (3,39) for example, using a 1000-L fixed-bed bioreactor, 42.2 kg L-malic acid per hour was produced continuously for 6 months. 

Examples of the use of immobilized enzymes in food processing and analysis have been listed by Olson and Richardson (1974) and Hultin (1983). L-aspartic acid and L-malic acid are produced by using enzymes contained in whole microorganisms that are immobilized in a polyacrylamide gel. The enzyme aspartase from Escherichia coli is used for the production of aspartic acid. Fumarase from Brevibacterium ammoni-agenes is used for L-malic acid production. 

The ( )-threo-[ >-2 H]malic acid showed a coupling constant of 4.4 0.2 Hz for the two non-equivalent protons. This was quite different from the value of J = 7.1-7.3 Hz for the [3-2H]malic acid obtained from the action of fumarase in 2H20. Hence the stereochemistry of fumarase was assigned as anti, 39 - 88. It should be noted that the coupling constants for the threo and erythro isomers are in accordance with the generalization, Jlrans >Jgauche. 

A combination of enzymatic and chemical methods was used to establish configuration for [2-2H]succinic acid [98], (25,3/ )-[3-2H]Malic acid was prepared by use of fumarase, and chemical methods (OH - Cl - H) were used to remove the chirality at position 2. 

The proof just given made no assumptions as to actual configurations. It can also be stated starting from the knowledge that fumarase hydrates fumaric acid to L-malic acid by anti addition on the Si-Si face. Hence, if the reaction is carried out in 2H20, the product is e/yf/iro-L-[3-2H]malic acid with (S) configuration at C-2, and (R) at C-3, 88. Hence, the sequence of reactions just discussed can be represented as follows ... 

If [2,3-2H2]fumarate is used as substrate for the fumarase reaction in HzO, the malic acid has (5) configuration at C-3, 123 in this case, the proton lost by action of aconitase is H not 2H, and 2H is finally removed by action of isocitrate dehydrogenase. Using normal projection formulae, this sequence is as follows ... 

It is also possible to convert nonchiral readily available industrial organic chemicals into valuable chiral natural-analogue products. This is demonstrated by the conversion of achiral fumaric acid to L(-)-malic acid with fumarase as the active enzyme. The same compound is converted to the amino acid L(-h)-aspartic acid by Escherichia bacteria that contain the enzyme aspartase. If pseudomonas bacteria are added, another amino acid L-alanine is formed (Eq. 9.10). 

The photochemical carboxylation of pyruvic acid by this process is endergonic by about AG° = 11.5 kcal mol and represents a true uphill photosynthetic pathway. The carbon dioxide fixation product can then act as the source substrate for subsequent biocatalyzed transformations. For example, photogenerated malic acid can act as the source substrate for aspartic acid (Figure 35). In this case, malic acid is dehydrated by fumarase (Fum) and the intermediate fumaric acid is aminated in the presence of aspartase (Asp) to give aspartic acid. 

The enantioselective addition of water to fumaric acid gives malic acid [118]. This highly efficient biocatalytic reaction is catalyzed by a fumarase. Although substrate tolerance of this enzyme is narrow, high enantioselectivities are obtained. 

L-Malic acid by water addition to fumaric acid Fumarase (Brevibacterium)... 

Catalysts also exhibit selectivity in their initial binding to reactants. Enzymes are well known for their ability to bind selectively to only one member of a pair of stereoisomers. The bound stereoisomer will undergo reaction, and the remaining isomer is inert to the reaction conditions. For example, the enantioselective addition of water to fumaric acid (the E isomer), which yields (.S )-malic acid (equation 9.7), is catalyzed by an enzyme called fumarase. Isomeric maleic acid (the Z isomer) fails to react in the presence of fumarase. 

Several industrial processes using lyases as catalysts have been reported. Perhaps the most prominent lyase-catalyzed process is the production of acrylamide from acrylnitrile. This process is carried out by the Nitto Chemical Company of Japan at a scale of more than 40,000 tons per year. Another example is the use of a fumarase for the production of (5 )-malic acid from fumaric acid. As shown in Fig. 7, a water molecule is added to the double bond in fumarate by means of an addition reaction. The result is a cleavage of the carbon-carbon double bond, and a formation of a new carbon-oxygen bond. A third example is bio-catalytic production of a cyanohydrin from a ketone. This reaction is catalyzed by a lyase called oxynitrilase. It consists of the cleavage of one carbon-oxygen bond, and the addition of a HCN molecule. The chirality of the product is based on the form of the enzyme used (/ -oxynitrilase or 5-oxynitrilase). ... 

Battat et al. [87] used A. flavus ATCC 13697 as the biocatalyst for the production of malic acid from glucose in a 16-1 stirred-tank fermentor. The optimal fermentation conditions are as follows agitation rate, 350 rpm Fe +, 12 mg/1 nitrogen (as ammonium sulfate), 271 mg/1 phosphate, 1.5 mM. Total amount of CaCOj added was 90 g/1. Fermentation was carried out at 32 °C for up to 200 h. Under the aforementioned conditions, 113 g/1 of L-malic acid were produced from 120 g/1 glucose utilized with an overall productivity of 0.59 g/l/h. Based on the molar yield, it was 128% for mahc acid and 155% for total acid (malic, fumaric and succinic acid). The increase in acid accumulation during the course of incubation coincides with the increase in the activities of NAD -malate dehydrogenase, fumarase and citrate synthase. 

L-Malic acid can also be produced from glucose using a combination of a fumaric acid producer Rhizopus arrhizus) and an organism with a high fumarase activity in the same fermentor [89,90]. 

The Mitsubishi Chemical Company has described a process for the commercial production of L-aspartate using an cx-amino-zr-butyric acid resistant mutant of B. flavum [11]. The enzyme is moderately thermal resistant, allowing the process to be run at 45°C. The process is run using immobilized cells in a fed batch system in which the biocatalyst is recycled [4]. An initial problem was the conversion of fumarate to malic acid by an intracellular fumarase activity, which led to low l-aspartic acid yields during the first cycle. This problem was circumvented by preheating the biocatalyst for 1 hour at 45°C, which completely destroyed the fumarase activity [4,11]. Recently, the aspartase gene from B. flavum has been cloned [28] and has presumably been used to improve the efficiency of this process. 

Finally, succinic acid is converted to oxaloacetic acid by way of fumaric and malic acids, the reactions being catalysed by succinic dehydrogenase, fumarase and malic dehydrogenase. One molecule of reduced flavoprotein is formed at the succinic dehydrogenase catalysed step, one of NADH2 by malic dehydrogenase (23) ... 

C—0 bond formation, L-malic acid from fumaric acid with fumarase 2000 t/y Amino GmbH, Tanabe Seiyaku [15]... 

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