Hydratases fumarate hydratase

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. 

Figure 9.2 Summary of reactions of the Krebs cycle. The names of the enzymes are dtrate synthase, aconitase, isodtrate dehydrogenase (there are two enzymes, one ubTizes NAD as the cofactor, the other NADPT it is assumed that the NAD -specific enzyme is that involved in the cycle), oxoglutarate dehydrogenase, sucdnyl CoA synthetase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase.

Water is now added to the double bond of fumarate by fumarate hydratase ( fuma-rase"), and chiral (2Sj-malate is produced. 

Carbonate dehydratase [Zn " ]— carbonic anhydrase Fumarate hydratase— fumarase ... 

Effect of Solvent on Arrhenius Plots. If water is a substrate, then the presence of an organic solvent, which may disrupt the structure and/or orientation of water, may alter the Arrhenius plot. For example, a linear plot is seen with fumarate hydratase in the presence of 10% methanol. However, the plot is biphasic in the presence of 10% ethanol . See Boltzmann Distribution Collision Theory Temperature Dependency, Transition-State Theory Energy of Activation On... 

This hydratase [EC 4.2.1.2] catalyzes the reversible dehydration of (5)-malate to form fumarate and water. 

This enzyme [EC 4.2.1.34], also known as mesaconase and mesaconate hydratase, catalyzes the conversion of (5 )-2-methylmalate to 2-methylfumarate and water. The enzyme will also catalyze the hydration of fumarate to (5 )-malate. 

This process has been operated successfully by the Tanabe Seiyaku Co. in Japan since 1973. Similar processes have since been commercialised by other companies, such as the Kyowa Hakko Co., often using different immobilisation methods such as polyurethane. The same iimnobilized cell approach has also been used by Tanabe since 1974 in their coimnercial process for the production of L-malic acid from fumarate using the hydratase activity of Brevibacterium ammoniagenes cells. 

The malate, oxaloacetate, and fumarate analogues 3-arsonolactate (86), 3-arsonopyruvate (68), and CE)-3-arsonoacrylate (87,88) have been made Ali and Dixon (88) found that the fumarate and malate analogues were not substrates for fumarate hydratase, but competitive inhibitors, arsonoacrylate, H203As—CH=CH—COOH, with fumarate ( / 1.8) and arsonolactate, H203As—CH2—CHOH—COOH, with malate (KJKm 1.6). Incidentally, although phosphonopyruvate is a poor substrate for malate dehydrogenase (89,90), 3-(hydroxyphosphinoyl)pyru-vate, HO—P(H)(0)—CH2—CO—COOH, is much better (89). It proved impossible to show the reverse reaction with arsonolactate (89, 91). 

Fumarate is hydrated to malate in a freely reversible reaction cat alyzed by fumarase (also called fumarate hydratase, see Figure 9.6). [Note- Fumarate is also produced by the urea cycle (see p. 251), in purine synthesis (see p. 293), and during catabolism of the amino acids, phenylalanine and tyrosine (see p. 261).]... 

Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and ATP (or GTP). This is an example of substrate-level phosphory lation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. The enzyme is inhibited by oxaloacetate. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to oxaloacetate by malate dehy drogenase, producing NADH. 

Fumarate hydratase (fumarase), which is discussed in Chapter 13, catalyzes the reversible hydration of fumaric acid to malic acid (Eq. 13-11). It was one of the first enzymes whose pH dependence was studied intensively. A bell-shaped pH dependence... 

Another example is provided by malic acid, a chiral molecule which also contains a prochiral center (see Eq. 9-74). In this case replacement of the pro-R or pro-S hydrogen atom by another atom or group would yield a pair of diastereoisomers rather than enantiomers. Therefore, these hydrogen atoms are diastereotopic. When L-malic acid is dehydrated by fumarate hydratase (Chapter 13) the hydrogen in the pro-R position is removed but that in the pro-S position is not touched. This can be demonstrated by allowing the dehydration product, fumarate, to be hydrated to malate in 2HzO (Eq. 9-74). The malate formed contains deuterium in the pro-R position. If this malate is now isolated and placed with another portion of enzyme in H20, the deuterium is removed cleanly. The fumarate produced contains no deuterium. 

The pH dependence of the action of fumarate hydratase indicates participation of both an acidic and a basic group with pfCa values of 5.8 and 7.1.56 See Chapter 9 for additional information. However, either anion or carbocation mechanisms might be possible. That the cleavage of the C-H bond is not rate limiting is suggested by the observation that malate containing 2H in the pro-R position is dehydrated at the same rate as ordinary malate. If the anion mechanism (Eq. 13-13) is correct, the 2H from the pro-R position of specifically labeled malate might be removed rapidly, while the loss of OH could be slower. If so, the 2H would be "washed out" of L-malate faster than could happen by conversion to fumarate followed by rehydration to malate. In fact, the opposite was observed. 

However, it has become clear that protons removed from a substrate to a basic group in a protein need not exchange rapidly with solvent (see Eq. 9-102). In fact, the proton removed by fumarate hydratase from malate is held by the enzyme for relatively long periods of time. Its rate of exchange between malate and solvent is slower than the exchange of a bound fumarate ion on the enzyme surface with another substrate molecule from the medium.59 Thus, the overall rate is determined by the speed of dissociation of products from the enzyme and we cannot yet decide whether removal of a proton precedes or follows loss of OH. ... 

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... 

As with fumarate hydratase, the enzyme holds the abstracted proton (for up to 7 x 10 5 s) long enough so that a ds-aconitate molecule sometimes diffuses from the enzyme and (if excess ds-aconitate is present) is replaced by another. The result is that the new ds-aconitate molecule sometimes receives the proton (intermolecular proton transfer). The proton removed... 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

Many addition and elimination reactions, e.g., the hydration of aldehydes and ketones, and reactions catalyzed by lyases such as fumarate hydratase are strictly reversible. However, biosynthetic sequences are often nearly irreversible because of the elimination of inorganic phosphate or pyrophosphate ions. Both of these ions occur in low concentrations within cells so that the reverse reaction does not tend to take place. In decarboxylative eliminations, carbon dioxide is produced and reversal becomes unlikely because of the high stability of C02. Further irreversibility is introduced when the major product is an aromatic ring, as in the formation of phenylpyruvate. 

The second substrate glyoxylate approaches from the other side of the molecule and condenses as is shown. Since any one of the three protons in either R or S chiral acetyl-CoA might have been abstracted by base B, several possible combinations of isotopes are possible in the L-malate formed. One of the results of the experiment using chiral (R) acetyl-CoA is illustrated in Eq. 13-43. The reader can easily tabulate the results of removal of the 2H or 3H. However, notice that if the base -B removes 2H (D) or 3H (T) the reaction will be much slower because of the kinetic isotope effects which are expected to be Hk/"k 7 and "k/ k = 16. A second important fact is that the pro-R hydrogen at C-3 in malate is specifically exchanged out into water by the action of fumarate hydratase. From the distribution of tritium in the malate and fumarate formed using the two chiral acetates, the inversion by malate synthase was established. See Kyte231 for a detailed discussion. 

Fumarase. See Fumarate hydratase Fumarase-aspartase family 685 Fumarate 481s, 516s, 683s Fumarate hydratase (fumarase) 526, 683,688 acid-base catalysis 471 concerted reaction 685 Fumarase A 688 Fumarase B 688 Fumarase C 683 mechanism 683 - 685 pH dependence 684 rates of substrate exchange 684 turnover number of 683 Fumarate reductase 785 Fumarylpyruvate 690s Function of state R 476 Fungal infections 20 Fungi 20... 

Second order reactions 458 Secondary kinetic isotope effect 592, 600 on fumarate hydratase 684 Secondary plots for kinetics of multisubstrate enzymes 465 Secondary structure 63... 

By application of the CIP rules the order of priority of the atoms directly attached to the chirality centre is O > C(0,0,(0)) > C(C,H,H) > H. The atom or group of lowest priority, hydrogen in this case, is already oriented away from the observer. Therefore the sequence of the remaining three groups can be directly deduced from the formula, and these are easily seen to be arranged in a counter-clockwise sense to the observer. It therefore follows that the formula represents (S)-2-hydroxysuccinic acid (formerly known as L-malic acid). The compound is produced in the citric acid cycle from fumaric acid by fumarate-hydratase (fumarase). 

Step 7 is the reversible hydration of fumarate to form malate, catalyzed by fumarate hydratase (which is usually called fumarase). 

A similar process is also used for the production of L-malic acid from fumarate, in this case using a hydratase enzyme derived from Brevibacterium ammoniagenes. Another variation of the Tanabe technology involves the synthesis of L-alanine from L-aspartic acid through the use of immobilized whole cells (P dacunae) containing aspartate-decarboxylase. 

After comparing the protein profiles of myocardial mitochondria between a chronic restraint stress group and a control group, 11 protein spots were found to change, of which seven were identified. Five of these proteins, carnitine palmitoyltransferase 2, mitochondrial acyl-CoA thioesterase 1, isocitrate dehydrogenase 3 (NAD ) alpha, fumarate hydratase 1, and pyruvate dehydrogenase beta, were foimd to decrease in abimdance following chronic restraint stress with fimctional roles in the Krebs cycle and lipid metabolism in mitochondria. The other two proteins, creatine kinase and prohibitin, increased after chronic restraint stress (liu et ak, 2004). 

---