Fumarase stereospecificity

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 ... 

Fumarate is hydrated in a stereospecific reaction by fumarase to give L-malate (Figure 20.17). The reaction involves fraw5-addition of the elements of water across the double bond. Recall that aconitase carries out a similar reaction. 

S. Except for oxido-reductases, transferases, and hydrolases, most ligases (enzymes that catalyze bond formation) are entirely substrate specific. Thus, fumarate hydratase (or fumarase) reversibly and stereospecifically adds water to fumaric acid to produce (S)-( — )-malic acid only (8) (Figure 1), and another enzyme, mesaconase, adds water to mesaconic acid to form (+ )-citramalic acid (9) (Figure 2). Although no extensive studies are available, it appears that neither fumarase nor mesaconase will add water stereospecifically to any other a,(3-unsaturated acid. 

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 ... 

The addition of water to fiimaric acid catalysed by fumarase is a highly stereospecific reaction and malic acid is formed as the sole product (Figure 2.22, X=H). The ammonia lyase 3-methylaspartase catalyses the similar addition of ammonia to yield L-aspartic acid. When uimatural substrates are used in these reactions (X =/= H), less success is experienced. An increasing X-group gives slow reaction rates. 

This enzyme is highly stereospecific it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate). In the reverse direction (from L-malate to fumarate), fumarase is equally stereospecific D-malate is not a substrate. 

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. 

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. 

The next step is the hydration of fumarate to form 1-malate. Fumarase catalyzes a stereospecific trans addition of a hydrogen atom and a hydroxyl group. The hydroxyl group adds to only one side of the double bond of fumarate hence, only the 1 isomer of malate is formed. 

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). 

Fumarate hydratase. The most studied enzyme of this group is probably the porcine mitochondrial fumarate hydratase (fumarase see also Chapter 9), a tetramer of 48.5-kDa subunits with a turnover number of 2 x 10 s T It accelerates the hydration reaction more than lO -fold. A similar enzyme, the 467-residue fumarase C whose three-dimensional structure is known, is foxmd in cells of E. coli when grown aerobically. The product of the fumarate hydratase reaction is L-malate (S-malate). The stereospecificity is extremely high. If the reaction is carried out in HjO an atom of H is incorporated into the pro-R position, i.e., the proton is added strictly from the re face of the trigonal carbon (Eq. 13-12). To obtain L-malate the hydroxyl must have been added from the opposite side of the double bond. Such anti (trans) addition is much more common in both nonenzymatic and enzymatic reactions than is addition of both H and OH (or -Y) from the same side (syn, cis, or adjacent addition). For concerted addition it is a natural result of stereoelectronic control. Almost all enzymatic addition and elimination reactions involving free carboxylic acids are anti with the proton entering from the re face. 

Aconitate dehydratase, succinate dehydrogenase, and fumarase yield stereospecific products. Labeling experiments with in methyl and carboxyl carbons of acetyl-CoA or in all of the carbons of oxaloacetate yield the following results in terms of the intermediates or product formed. 

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

Enzyme-catalyzed reactions are also stereospecific—an enzyme typically catalyzes the reaction of only one stereoisomer. For example, fumarase catalyzes the addition of water to fumarate (the trans isomer) but not to maleate (the cis isomer). 

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... 

Fumarase [EC 4.2.1.2] and malease [EC 4.2.1.31] catalyze the stereospecific addition of water onto carbon-carbon double bonds conjugated with a carboxylic acid [1570]. The analogous addition of ammonia is catalyzed by aspartase [EC 4.3.1.1], 3-methylaspartase [1571], and phenylalamine ammonia lyase [EC 4.3.1.5],... 

Two of the enzymes of the tricarboxylic acid cycle, aconitase and fumarase, catalyze reactions in which water is added reversibly to an unsaturated polycarboxylic acid. Both enzymes exhibit rigid stereospecificity fumarase forms only L-malate from fumarate and forms only fumarate (trans) and not maleate (czs-ethylenedicarboxylic acid), and aconitase reacts with only cis-, not imns-aconitate, and with D-, not L-isocitrate. Citrate is a symmetrical molecule, with no optical isomers, but it will be shown that steric factors also enter into the reaction of this substrate with aconitase. The enzymes of the tricarboxylic acid cycle, in contrast to the glycolytic enzymes, are associated with intracellular granules known as mitochondria. Studies of the individual enzymes have depended to a large extent on the separation of soluble activities from these particles. Aconitase and fumarase are released from the particles very rapidly under mild conditions often in the preparation of cell-free homogenates these activities are largely solubilized, and special care must be taken to demonstrate their origin in mitochondria. 

Hydration of alkenes occurs stereospecifically in biological systems. Hydration of fumarate by D2O catalyzed by fumarase yields (2S,3i )-3-deuteriomalate. Determine the stereochemistry of addition. 

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