Fumarase and

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. 

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. 

Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes citrate synthase, aconitase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. 

Fig. 25-15). In every case it is NH3 or an amine, rather than an OH group, that is eliminated. However, the mechanisms probably resemble that of fumarate hydratase. Sequence analysis indicated that all of these enzymes belong to a single fumarase-aspartase family.64 65 The three-dimensional structure of aspartate ammonia-lyase resembles that of fumarate hydratase, but the catalytic site lacks the essential HI 88 of fumarate hydratase. However, the pKa values deduced from the pH dependence of Vmax are similar to those for fumarase.64 3-Methylaspartate lyase catalyzes the same kind of reaction to produce ammonia plus czs-mesaconate.63 Its sequence is not related to that of fumarase and it may contain a dehydroalanine residue (Chapter 14).66... 

In the Briggs-Maldane mechanism, when k2 is much greater than k-i, kcJKM is equal to kx, the rate constant for the association of enzyme and substrate. It is shown in Chapter 4 that association rate constants should be on the order of 108 s l M l. This leads to a diagnostic test for the Briggs-Haldane mechanism the value of kaJKu is about 107 to 108 s-1 M-1. Catalase, acetylcholinesterase, carbonic anhydrase, crotonase, fumarase, and triosephosphate isomerase all exhibit Briggs-Haldane kinetics by this criterion (see Chapter 4, Table 4.4). 

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. 

To understand how the TCA cycle responds kinetically to changes in demand, we can examine the predictions in time-dependent reaction fluxes in response to changes in the primary controlling variable NAD. Figure 6.4 plots predicted reaction fluxes for pyruvate dehydrogenase, aconitase, fumarase, and malate dehydrogenase in response to an instantaneous change in NAD. The initial steady state is obtained... 

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. 

Following are a set of assay conditions for marker enzymes of mitochondria (citrate synthetase, malic dehydrogenase, fumarase, and succinate dehydrogenase) and glyoxysomes (citrate synthetase, malate synthetase, and malic dehydrogenase). Some or all of these activities may be assayed across the density gradient. Their quantitative distribution is shown in Table 9-2. 

In contrast with the fall in the activity of muscle glycolytic enzymes in human muscular dystrophy, Dreyfus and his colleagues (DIO) found little or no decrease in the concentrations of certain enzymes involved in oxidative breakdown of fuel, notably succinate dehydrogenase, cytochrome oxidase, fumarase, and aconitase. In the mouse myopathy, the concentration of cytochrome oxidase is increased (W12) elevated levels of respiratory enzymes have been reported also in myopathy resulting from vitamin E deficiency (D6) and in genetically dystrophic chickens... 

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. 

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) ... 

Pancreatic ribonuclease is markedly inhibited by heparinoids but not by mucopolysaccharides, with sulphated polyvinyl alcohol >sulphated corn amylosOsulphated cellulose > sulphated corn amylopectin> sulphated dextran, sulphated pectic acid, polyvinyl sulphonate with 2 T 7 per cent sulphur>sulphated nitro chitin, sulphated nitro chitosan and heparin . Single injections of heparin in mice result in significant inhibition of the acid alkaline ribonuclease of the liver after the injection. This probably explains the accumulation of ribonucleic acid in tissue culture cells in the presence of heparin. Heparin also inhibits rat acid phosphatase, glucuronidase , catalase , fumarase and elastase. 

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