Fumarase activity

I Takata, K Kayashima, T Tosa, I Chibata. Improvement of stability of fumarase activity of Brevibacterium flavum by immobilization with A>carrageenan. J Ferment Technol 60 431-437, 1982. 

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. 

Two trends in this table are noteworthy. The first is the decreased flux of label through the "straight pathways (ST) in the presence of ethanol. This trend indicates that ethanol markedly increases the scrambling of intermediates by fumarase activity as noted also by Cohen al. (16) in isolated hepto-cytes. In addition, ethanol also decreases the relative flux of label through the TCA cycle. This observation suggests that the... 

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 ... 

Pierpoint, W.S. Mitochondrial preparations fiom the leaves of tobacco Nicotiana tabacum). 3. GlycoUic oxidase and fumarase activity Biochem. J. 75 (1960) 511-515. Pierpoint, W.S. Mitochondrial preparations from the leaves of tobacco Nicotiana tabacum). 4. Separation of some components by density-gradient centrifuging Biochem. J. 82 (1962) 143-148. 

Which would you expect to have a greater effect on the rate of urea biosynthesis, a defect in fumarase activity or a defect in alanine aminotransferase ... 

Enrichment at specific carbon atoms of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, 3-hydroxybutyrate and lactate measured. Alanine enters TCA cycle predominately through pyruvate car-boxylation in presence of ethanol which enters as acetyl CoA. Pentose cycle and fumarase activities estimated. Glutamine not in isotopic equilibrium with glutamate. 

Active immobilized cells investigation of 660 kinetics and decay of fumarase activity of the immobilized cells... 

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]. 

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