Fumarase reaction catalyzed

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). 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

In the second step of the /3-oxidation cycle (Fig. 17-8a), water is added to the double bond of the tran.s-A2-enoyl-CoA to form the l stereoisomer of /3-hydroxyacyl-CoA (3-hydroxyacyl-CoA). This reaction, catalyzed by enoyl-CoA hydratase, is formally analogous to the fumarase reaction in the citric acid cycle, in which H20 adds across an a-/3 double bond (p. XXX). 

As with fumarase, the reactions catalyzed by serine dehydratase and dihydroxyacid dehydratase do not require release and rebinding of intermediate. The reaction mechanisms should be describable as adapted versions of one-half only of Figure 3. 

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. 

Fumarate is hydrated. Fumarate is converted to L-malate in a reversible stereospecific hydration reaction catalyzed by fumarase (also referred to as fumarate hydratase) ... 

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. 

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... 

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

Equilibria of Aconitase and Fumarase. The reactions catalyzed by fumarase and aconitase have small free-energy changes. Omitting water from the calculation, the equilibrium constant for fumarase, K, = (malate)/(fumarate) = about 3-4 at room temperature and pH 7. This reaction is temperature dependent, forming relatively more fumarate at higher temperatures. The aconitase equilibrium must include two separate reactions the equilibration of cfs-aconitate with... 

A similar pathway for the formation of sucdnate in both Actinobadllus sp. 130Z and A. sucdniciproducens has been proposed. The formation of oxalacetate fi-om PEP via CO2 fixation is the first key step. The enzymes malate dehydrogenase, fumarase, and fumarate reductase, all enzymes of the tricarboxylic acid (TCA) cycle, work in a reductive fashion toward succinate (Van der Werf et al. 1997 Samuelov et al. 1991). The reactions catalyzed are as follows ... 

Biochemical processes are catalyzed by enzymes that have multiple stereogenic centers and are therefore chiral. Enzymes provide a chiral environment in which to form stereogenic centers. As a consequence, only one enantiomer forms firom an enzyme-catalyzed reaction, even if the reactant is achiral. For example, fumaric acid reacts with water in an addition reaction catalyzed by the enzyme fumarase in the citric acid cycle to give only (i)-malic acid. We show the carboxyhc acids as their conjugate bases because they are ionized at pH 7. These ionic compounds are called fumarate and malate. This reaction converts fumarate to (i)-malate. 

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 ... 

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... 

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. 

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. 

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. 

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 MAD" " to give oxaloacetate. The addition is catalyzed by fumarase and is mechanistically similar to the addition of water to d.s-aconitate in stejr 2. The reaction occurs through an enolate-ion intermediate, which is protonated on the side opposite the OH, leading to a net anti addition. 

Enzyme-catalyzed reactions are usually much more chemically selective than their laboratory counterparts. Fumarase, for example, is completely inert toward maleic acid, the cis isomer of fumaric acid. Nevertheless, the fundamental processes of oi nic chemistry are the same in the living cell and in the laboratory. 

The enantioselective addition of water to fumaric acid gives malic acid [118]. This highly efficient biocatalytic reaction is catalyzed by a fumarase. Although substrate tolerance of this enzyme is narrow, high enantioselectivities are obtained. 

Catalysts also exhibit selectivity in their initial binding to reactants. Enzymes are well known for their ability to bind selectively to only one member of a pair of stereoisomers. The bound stereoisomer will undergo reaction, and the remaining isomer is inert to the reaction conditions. For example, the enantioselective addition of water to fumaric acid (the E isomer), which yields (.S )-malic acid (equation 9.7), is catalyzed by an enzyme called fumarase. Isomeric maleic acid (the Z isomer) fails to react in the presence of fumarase. 

Several industrial processes using lyases as catalysts have been reported. Perhaps the most prominent lyase-catalyzed process is the production of acrylamide from acrylnitrile. This process is carried out by the Nitto Chemical Company of Japan at a scale of more than 40,000 tons per year. Another example is the use of a fumarase for the production of (5 )-malic acid from fumaric acid. As shown in Fig. 7, a water molecule is added to the double bond in fumarate by means of an addition reaction. The result is a cleavage of the carbon-carbon double bond, and a formation of a new carbon-oxygen bond. A third example is bio-catalytic production of a cyanohydrin from a ketone. This reaction is catalyzed by a lyase called oxynitrilase. It consists of the cleavage of one carbon-oxygen bond, and the addition of a HCN molecule. The chirality of the product is based on the form of the enzyme used (/ -oxynitrilase or 5-oxynitrilase). ... 

Fumarase is an enzyme component of the TCA cycle that catalyzes the reversible reaction of fumarate to L-malate with equilibrium favoring malate production. It is a soluble enzyme with high turnover number. In one report, fumarate content in some organisms can be as high as lOOOmg/kg of wet cells [80]. Theoretically, a malate weight yield of 115% can be obtained from fumarate. However, in reality, a weight yield of 90-95% is often obtained. 

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