Enzyme fumarase

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 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. 

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 ... 

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. 

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). 

Hydration and dehydration reactions are common in biological pathways. The enzyme fumarase catalyzes the reversible addition of waterto the double bond of fumaratetoform malate. In contrast to the harsh conditions used in the chemical reaction, the enzymatic reaction takes place at neutral pH and at 37 °C. 

Generally speaking, these distinctions have not been observed by biochemists. Stereoselective has been little used, and stereospecific has been used to cover almost all aspects of the impact of stereochemical influences on reactions in living tissues or enzyme systems. Consider, for instance, the enzymatic hydration of fumarate by the enzyme, fumarase. Since there is a relationship between the structure of the substrate and product, the process could be described as stereospecific. Yet the definition of stereospecific requires that it be shown that the isomer of fumaric acid gives rise to a product which is stereochemically different from L-malate. Since the enzyme, however, does not catalyze any reaction with the (Z)-isomer (maleic acid) it is not clear whether stereospecific actually applies. 

Biological organic chemistry takes place in the aqueous medium inside cells rather than in organic solvents, and it involves complex catalysts called enzymes. Nevertheless, the kinds of biological roactions that occur are remarkably nimiUr to laboratory reactions. Thus, there are many cases of biological addition reactions to alkenes. For example, Che enzyme fumarase... 

STEPS 7-8 Regeneration of oxaloacetate. Catalyzed by the enzyme fumarase. conjugate miclcophilic addition of w ater to fumarate yields t-malate in a reaction simUar to tliat of step 2 in the fatty acid j3>oxidation pathway. Oxida-i m with NAD then, gives oxaloacetate in a step catalyzed by malate dehydrogenase, and the citric acid cycle has return to ita starting point, ready to revolve again. 

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. 

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). 

Similarly, the absolute stereochemistry of the conversion of fu-marate to malate by the enzyme fumarase was confirmed by a neutron-diffraction structure determination of the structure of the (- -)-i -a-methylbenzylammonium salt of malate (see Figure 14.6). The malate was prepared enzymatically with the use of heavy water (D2O) so that... 

FIGURE 14.16. Stereochemistry of the enzymatic conversion of fumarate to malate (Ref. 56). Shown are (a) the crystal structure of the complex, and (b) the deduced steric course of the reaction catalyzed by the enzyme fumarase. 

At pH 6, the enzyme fumarase (50 kDa/subunit) converts fumarate to malate, and has a soluble Km for fumarate of 5.7 pM. The dissociation rate constant for the enzyme-substrate complex into enzyme plus malate has been measured as 1.45 x lO -1. 

In optimum conditions, one molecule of the enzyme fumarase can convett 8x10 molecules pet minute of fumarate to malate. How many molecules could be converted by one of these enzyme molecules in 30 days ... 

Cell fractionation by mechanical rupture has already come under investigation. Two separate studies of mechanical rupture of yeast showed different rates of release for enzymes in different cell locations (13,14). Wall-linked and periplasmic enzymes were released relatively faster than total protein, soluble cytoplasmic enzymes at about the same rate, and the mitochondrial enzyme fumarase later than total protein (13). Proteolysis by the yeast s own enzymes was not found to be a problem. Activities of the released enzymes declined slowly or not at all when disruption was continued after the end of protein release, and the effect of shear was not separated from the effect of proteolysis. Shetty and Kinsella (15) also found a low rate of proteolysis after mechanical disruption, though thiol reagents added to weaken the cell walls before disruption caused an important increase in the extent of protein breakdown. 

The organic reactions that occur in biological systems are catalyzed by enzymes. Enzyme-catalyzed reactions are almost always completely stereoselective. In other words, enzymes catalyze reactions that form only a single stereoisomer. For example, the enzyme fumarase, which catalyzes the addition of water to fumarate, forms only... 

When fumarate reacts with D2O in the presence of the enzyme fumarase, only one isomer of the product is formed. Its structure is shown. Is the enzyme catalyzing a syn or an anti addition of D2O ... 

Dehydration reactions are known to occur in many important biological processes. Instead of being catalyzed by strong acids, which would not be available to a cell, they are catalyzed by enzymes. Fumarase, for example, is an enzyme that catalyzes the dehydration of malate in the... 

Figure 11 The neutron diffraction results of the enzymatic reaction of Scheme 3. The known absolute configuration of the (+)(R)a-phenylethylammonium cation (a) showed that the product malate anion, produced enzymatically by the enzyme fumarase (b), had the absolute configuration (—)(2S,3R), meaning that the enzymatic addition of D2O was stereospecifically trans. (Published in Bau, Brewer, Chiang, Fujita, Hoffman, Watkins and Koetzle, Elsevier 1983)...

We have recently provided a stereospecific synthesis of samples of D-serine that are stereospecifically labeled at C-3 (94) (Scheme 23). This relies on the known (82) stereospecificity of the enzyme fumarase (EC 4.2.1.2) which adds water across the double bond of fumaric acid 72 with anti stereospecificity to give (2S)-malic acid 83. Thus [ H2]fumaric acid, 72, = H, in H2O... 

Scheme 45). When samples of (3S)- and (3R)-[3- Hi]cysteine 134, and 134, Hg = H, respectively, prepared using 0-acetylserine sulfhydrase as above, were incubated with this synthase and the resultant samples of )S-cyanoalanine 144 were degraded to malates 83a via aspartates, the pro-R specific enzyme fumarase could be used to assess the stereochemistry, as shown in Scheme 45 (87). The P replacement 134 144 again occurred with... 

The turnover number of the enzyme fumarase that catalyzes the reaction,... 

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