Fumarate/fumarase activity

Fig. 5.9. Proposed scheme for the intramitochondrial metabolism of malate by Hymenolepis diminuta. Abbreviations ME, malic enzyme F, fumarase T, transhydrogenase FR, fumarate reductase ETS, electron transport system. Once within the matrix compartment, malate oxidation, as catalysed by malic enzyme, results in NADPH formation. Via the activity of fumarase, malate also is converted to fumarate in the matrix compartment. NADPH then serves as a substrate for the inner-membrane-associated transhydrogenase and transhydrogenation between NADPH and matrix NAD is a scalar reaction associated with the matrix side of the inner membrane. Matrix NADH so formed reduces the electron transport system via a site on the matrix side of the inner membrane permitting fumarate reductase activity. The reduction of fumarate to succinate results in succinate accumulation within the matrix compartment. (After McKelvey Fioravanti, 1985.)...

The hydration of fumaric acid [( )-butenedioic acid, 1 R = H] to (S)-2-hydroxybutanedioic acid (2) is catalyzed by the enzyme fumarase. This reaction can be run even on an industrial scale, exploiting the fumarase activity of immobilized microorganisms77. Unfortunately, the substrate spectrum of fumarase is very narrow. Nevertheless, (Z)-2-chlorobutenedioic acid (3, R = Cl) could be diastereo- and enantioselectively hydrated to (2S,3/ )-2-chloro-3-hydroxybu-tanedioic acid (4) on a 50-gram scale, employing commercially available pig heart fumarase [EC 4.2.1.2.]78. 

C-IO) Fumarase deficiency. There is a deficit in the transformation of fumarate to malate. The infant has developmental retardation, with abnormal neuromuscular function, lactic acidemia, and fumarate aciduria. The lactic acidosis may result from a backup of Krebs cycle function, all the way to lactate. Lactic acidosis may also be present in rare disorders of cytochrome oxidase activity. Diagnostically, there is a deficit in fumarase activity in assay of liver and skeletal muscle mitochondria. 

The stereospecific addition of water to fumaric add catalyzed by the enzyme fumarase yields optically pure L-malic add (Fig. 28). A Brevibac-terium flctvum strain with high fumarase activity has been used industrially for the commerdal production of L-malic add (97). The substrate specificity of fumarase is narrow and hence its broader application in organic synthesis has been somewhat limited. However, it has been shown to synthesize L-fferco-chloromalic add in very high optical purity (98). 

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. 

In 1974 we succeeded in the industrial production of L-malic acid from fumaric acid by Brevibacterium ananoniagenes cells immobilized by the polyacrylamide gel method [9, 10]. The asymmetric reaction catalyzed by the fumarase activity of the cells is shown below. 

A one-month-old baby boy was brought to the hospital showing severely delayed development and cerebral atrophy. Blood tests showed high levels of lactate and pyruvate. By three months of age, very high levels of succinate and fumarate were found in the urine. Fumarase activity was absent in the liver and muscle tissue. The baby died at five months of age. This was the first reported case of fumarase deficiency and the defect was recognized too late for effective therapy to be administered. What reaction is catalyzed by fumarase How would a deficiency of this mitochondrial enzyme account for the baby s symptoms and test results ... 

Additional suggestive evidence for the existence of a second distinct fumarase in this fungus comes from the analysis of fumarase activity in cell lysates. Fumarase in lysates of R. oryzae from medium B (growth medium) has a lower value for fumaric acid (0.78 mM) than for L-malic acid (2.9 mM), similar to fumarase from lysates of S. cerevisiae (Pines et al., 1996). Fumarase activities (with L-malic acid as the substrate) in both these lysates were not inhibited by fumaric acid. In sharp contrast, fumarase activity measured in extracts prepared from R. oryzae cells incubated in medium C (production medium), but not with S. cerevisiae, was completely inhibited by 2 mM fumaric acid (E. Battat and I. Goldberg, unpublished data cited in Goldberg et al., 2006). 

It should be mentioned that with another Rhizopus strain. Ding et al. (2011) showed in cell extracts, in accordance to previous findings that lowering the urea concentrations in the medium from 2.0 to 0.1 g/L caused an increase of 300% in the cytosolic fumarase activity, accompanied with an increase in fumaric acid production. 

Kinetics of Fumarase Activity. The kinetics of the fumarase reaction have been studied intensively by Alberty and his collaborators. They have found that interaction of enzyme with phosphate can cause activation at low phosphate concentrations, but that at high concentrations, phosphate acts as a competitive inhibitor. An unusual effect was noted when the effect of fumarate concentration on the rate of hydration was measured. At low substrate concentrations the Lineweaver-Burk plots are linear, but at higher concentrations the rate is faster than anticipated. This phenomenon was interpreted as indicating an interaction of fumarate with the enzyme at sites other than the catalytic site, to form a more active enzyme. At very high substrate concentrations (0.1 M) there is inhibition of the reaction, and the theoretical V— is never attained. 

In general, endogenous metabolism of anaerobic bacteria was found to be more stable, when biocatalysts based on immobilized cells of P. shermanii and E. coli were compared with respect to the reactions shown above (Ikonnikov, 1985). P. shermanii had a higher aspartase activity than P. pentosaceum, P. petersonii and P. technicum (Kalda and Vorobjeva, 1981). After 3 days of incubation with continuous stirring at 37°C and pH 8.5, the extent of substrate conversion (ammonium fumarate) was 95-96% and 75-90% in the case of E. coli K-12 and P. shermanii, respectively. In addition to aspartic acid, the reaction mixtures of the two strains also contained malic acid. Heat treatment of the biomass of P. shermanii (50 C, 1.5 h, pH 5.0) resulted in a complete inactivation of fumarase, while the activity of aspartase was retained (Kalda and Vorobjeva, 1980, 1981). As a result of the elimination of fumarase activity, the yield of L-aspartic acid from ammonium fumarate was increased up to 96-98% the incubation time was also shortened since no substrate was diverted to the side reaction forming malate. 

In addition two mentally retarded adults with fumaric aciduria have been described, but the abnormality was thought to be due to a renal fumaric acid resorption defect. Fumarase activity was not determined in these patients. 

Deficiency of fumarase causes marked elevations of fumaric acid and often other Krebs cycle intermediates in the urinary organic acids profile. The diagnosis is confirmed by measurement of fumarase activity in cultured skin fibroblasts, leukocytes or affected organs. For defining the carrier status of family members, fumarase activity measurement in blood mononuclear cells appears to be a good, easy screening tool [7-9]. Mutation analysis can confirm the results of enzyme studies [11]. 

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... 

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. 

Stevens and Stevens (1979) measured the hydration dependence of glucose-6-phosphate dehydrogenase, hexokinase, fumarate hydratase (fumarase), and glucose-6-phosphate isomerase (phosphoglucose isom-erase) over the range 0.1-0.6 h. Serum albumin was used as a carrier protein to buffer the water content. The hydration isotherms of the enzymes and the serum albumin were assumed to be similar. For the first three enzymes activity was detected (0.05% of full solution activity) near 0.2 h. Activity was measurable for the isomerase at 0.15 h. In all cases, even at 0.3 h, the activity in the powder was less than 5% of the solution rate. Diffusion of substrates in the powder may be rate limiting. The amount of albumin in the powder affected the rate. 

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). 

There is still a third possible mechanism for the fumarate hydratase reaction. The proton and hydroxyl groups may be added simultaneously in a concerted reaction. However, observed kinetic isotope effects are not consistent with this mechanism. In 1997 the structure of fumarase C of E. coli was reported. Each active site of the tetrameric enzyme is formed using side chains from three different subunits. The H188 imidazole is hydrogen bonded to an active site water molecule and is backed up by the E331 carboxy-late which forms a familiar catalytic pair. However, these results have not clarified the exact mode of substrate binding nor the details of the catalytic mechanism. Structural studies of fumarate hydratase from yeast and the pig are also in progress. 

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. 

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