Fumarase

Process development with Brevibacterium ammoniagenes (introduced in 1974), similarly to the development in the i-aspartate process led to 25-fold improvements, mainly through use of a Brevibacteriumflavum strain immobilized on ic-carrageenan gel with polyethyleneimine (PEI) crosslinker, so that a half life of 310 days at 37°C has been achieved. 

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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... 

The enzyme fumarase catalyzes the stereospecific addition of water to fumarate to form L-malate. A standard solution of fumarase, with a concentration of 0.150 tM, gave a rate of reaction of 2.00 tM mim under conditions in which the concentration of the substrate was significantly greater than K. The rate of reaction for a sample, under identical conditions, was found to be 1.15 tM mimh What is the concentration of fumarase in the sample ... 

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. 

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate (Figure 20.17). The reaction involves fraw5-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction. 

Glyoxysomes do not contain all the enzymes needed to run the glyoxylate cycle succinate dehydrogenase, fumarase, and malate dehydrogenase are absent. Consequently, glyoxysomes must cooperate with mitochondria to run their cycle (Figure 20.31). Succinate travels from the glyoxysomes to the mitochondria, where it is converted to oxaloacetate. Transamination to aspartate follows... 

Steps 7-8 of Figure 29.12 Hydration and Oxidation The final two steps in the citric acid cycle are the conjugate nucleophilic addition of water to fumarate to yield (S)-malate (L-malate) and the oxidation of (S)-malate by NAD+ to give oxaloacetate. The addition is cataiyzed by fumarase and is mechanistically similar to the addition of water to ris-aconitate in step 2. The reaction occurs through an enolate-ion intermediate, which is protonated on the side opposite the OH, leading to a net anti addition. 

Enzymes a) citrate synthase b) aconitase c) isocitrate dehydrogenase d) a-oxoglutarate dehydrogenase e) succiny CoA synthetase f) succinate dehydrogenase g) fumarase h) malate dehydrogenase i) nucleoside diphosphokinase. 

To prevent the formation of byproducts like L-malic add and D-alanine, die cells undergo a pH-treatment to inactive fumarase and alanine racemase. Several reactor conformations have been investigated, but a two reactor system was found to be the most effective. The flow sheet of this two reactor system is given in Figure A8.15. In the first reactor L-aspartic add is formed, which reacts in die second reactor to L-alanine. 

Fig. 28. Porcine Fumarase subject to shearing, with and without air/liquid interface, with a stainless steel disc at a mean velocity gradient of 6490 s at 30 °C. The enzyme was also sheared, with and without air/liquid interface, with a 6 bladed Rushton turbine at a mean velocity gradient of 22700 s at 30 °C [107]...

Marietta MA, K-F Chenng, C Walsh (1982) Stereochemical studies on the hydration of monofluorofumarate and 2,3-difluorofumarate by fumarase. Biochemistry 21 2637-2644. 

The activity of many enzymes is pH-dependent because the enzyme may ionize in solution and the biological activity of unionized and ionized forms may be different. In this case, the rate of an enzyme-mediated reaction can be expected to depend on the acidity of the solution. If the enzyme can lose more than one proton as the pH increases (Figure 8.12), the rate of reaction as a function of pH may display a maximum if the forms of the enzyme in strongly acidic or strongly basic solution are inactive, but the intermediate, monoanion, is active. An example of this behavior is provided by fumarase (Figure 8.13). 

The pKa values for the two ionizations of fumarase can be determined experimentally to be approximately 5.9 and 7.5, thus these figures provide an appropriate way to define what, in the context of this problem, is meant by "medium" pH (Table 8.3). 

Defects of the Krebs cycle. Fumarase deficiency was reported in children with mitochondrial encephalomyop-athy. Usually, there is developmental delay since early infancy, microcephaly, hypotonia and cerebral atrophy, with death in infancy or early childhood. The laboratory hallmark of the disease is the excretion of large amounts of fumaric acid and, to a lesser extent, succinic acid in the urine. The enzyme defect has been found in muscle, liver and cultured skin fibroblasts [16]. 

The criteria for gene displacement in this study were strict. The reactions catalyzed were required to have the same EC (Enzyme Commission) number, which implies that the same cofactors had to be involved. In the example of reactions involved in the citric acid cycle given previously, when only the carbohydrate substrate and product of the reaction were the same, we could identify gene displacements at 6 of the 11 steps included in the analysis. Only two of those (malate dehydrogenase and fumarase) met the criteria in Galperin et al. (1998). 

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. 

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