Fumarate reaction with fumarase

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. 

After equilibration with fumarase, the mixture of fumarate and malate is analyzed for 3H. If tritium is originally present in the pro-3S position, the equilibration will not remove it (150 plus 152). On the other hand, tritium in the pro-3R position will be lost to the water by way of 153. To facilitate the analysis, [14C]acetate is added initially and the 3H I4C ratio is determined on the malate produced by malate synthase, before and after incubation with fumarase. The tritium content of the incubation water is also determined. The % retention of 3H in the fumarase reaction is given the symbol, F [127]. In detailed analyses, it has been shown that chirally pure (7 (-acetate is characterized by F = 79 and (S (-acetate by F= 21 [129]. The F value actually depends on four factors ... 

Mitsubishi has also developed a process for production of D-aspartic acid (d-2) and L-malic acid (4) by incubation of racemic aspartic acid with the exclusively L-selective aspartase in combination with fumarase, thereby preventing the reaction going backwards by conversion of the generated fumaric acid into L-malic acid 4. ... 

Isotopic Studies with Fumarase. The kinetic data reported above have led to preliminary attempts to interpret the nature of the binding of substrate to enzyme in terms of specific amino acid side chains. Another approach to the nature of the reaction was made in studies with D20. It was found that the hydrogens of fumarate do not equilibrate with... 

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 ... 

A general type of chemical reaction between two compounds, A and B, such that there is a net reduction in bond multiplicity (e.g., addition of a compound across a carbon-carbon double bond such that the product has lost this 77-bond). An example is the hydration of a double bond, such as that observed in the conversion of fumarate to malate by fumarase. Addition reactions can also occur with strained ring structures that, in some respects, resemble double bonds (e.g., cyclopropyl derivatives or certain epoxides). A special case of a hydro-alkenyl addition is the conversion of 2,3-oxidosqualene to dammara-dienol or in the conversion of squalene to lanosterol. Reactions in which new moieties are linked to adjacent atoms (as is the case in the hydration of fumarate) are often referred to as 1,2-addition reactions. If the atoms that contain newly linked moieties are not adjacent (as is often the case with conjugated reactants), then the reaction is often referred to as a l,n-addition reaction in which n is the numbered atom distant from 1 (e.g., 1,4-addition reaction). In general, addition reactions can take place via electrophilic addition, nucleophilic addition, free-radical addition, or via simultaneous or pericycUc addition. 

Fig. 5.9. Proposed scheme for the intramitochondrial metabolism of malate by Hymenolepis diminuta. Abbreviations ME, malic enzyme F, fumarase T, transhydrogenase FR, fumarate reductase ETS, electron transport system. Once within the matrix compartment, malate oxidation, as catalysed by malic enzyme, results in NADPH formation. Via the activity of fumarase, malate also is converted to fumarate in the matrix compartment. NADPH then serves as a substrate for the inner-membrane-associated transhydrogenase and transhydrogenation between NADPH and matrix NAD is a scalar reaction associated with the matrix side of the inner membrane. Matrix NADH so formed reduces the electron transport system via a site on the matrix side of the inner membrane permitting fumarate reductase activity. The reduction of fumarate to succinate results in succinate accumulation within the matrix compartment. (After McKelvey Fioravanti, 1985.)...

The proof just given made no assumptions as to actual configurations. It can also be stated starting from the knowledge that fumarase hydrates fumaric acid to L-malic acid by anti addition on the Si-Si face. Hence, if the reaction is carried out in 2H20, the product is e/yf/iro-L-[3-2H]malic acid with (S) configuration at C-2, and (R) at C-3, 88. Hence, the sequence of reactions just discussed can be represented as follows ... 

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. 

Fumarase. Fumarase is determined by measuring the conversion to malate to fumarate. This assay was used in the opposite direction by Racker. The mixture contains, in a volume of 1.3 ml, 8.0 x 10 phosphate buffer (adjusted to pH 7.5), 4.0mM dithiothreitol, 8.0mM sodium malate, and 2 to 25 fxg of protein. The reaction is initiated with malate and is followed at 240 nm. The molar extinction coefficient of fumarate is 2.6 X lO cm /mole. 

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. 

There is still a third possible mechanism for the fumarate hydratase reaction. The proton and hydroxyl groups may be added simultaneously in a concerted reaction. However, observed kinetic isotope effects are not consistent with this mechanism. In 1997 the structure of fumarase C of E. coli was reported. Each active site of the tetrameric enzyme is formed using side chains from three different subunits. The H188 imidazole is hydrogen bonded to an active site water molecule and is backed up by the E331 carboxy-late which forms a familiar catalytic pair. However, these results have not clarified the exact mode of substrate binding nor the details of the catalytic mechanism. Structural studies of fumarate hydratase from yeast and the pig are also in progress. 

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. 

When a V/K profile is represented by Eq. (75) we cannot a priori tell whether the group with pK has to be ionized and the group with pK2 protonated, or vice versa, since the shape of the pH profile will be exactly the same. For example, fumarase (fumarate hydratase) has an imidazole (p/f 7.1) that must be protonated and a carboxyl group (p/f 5.85) that must be ionized for reaction of malate, whereas the imidazole must be neutral and the carboxyl protonated for reaction of fumarate (7S). It is common for such reverse protonation to be required in one direction of an enzymic reaction. 

Although the major route for aspartate degradation involves its conversion to oxaloacetate, carbons from aspartate can form fumarate in the urea cycle (see Chapter 38). This reaction generates cytosolic fumarate, which must be converted to malate (using cytoplasmic fumarase) for transport into the mitochondria for oxidative or anaplerotic purposes. An analogous sequence of reactions occurs in the purine nucleotide cycle. Aspartate reacts with inosine monophosphate (IMP) to... 

Obviously, the V profiles for the slow nonsticky substrate are the best choice, since these Vprofiles will show the correct piTa vcdues of amino acid side chains on enzyme in the enzyme-substrate complex that are responsible for catalysis. The apparent pK s of the catalytic groups of fumarase have been measured in both directions (Brandt et al, 1963). When malate is used as a substrate, Vprofile is beU-shaped showing two p a s, 6.4 and 9.0, respectively temperature variation of these groups indicates that p Ta 6.4 corresponds to dissociation of a carboxyl and pXa 9.0 to dissociation of a histidine side chain in the active site of enzyme. Thus, from the malate side of reaction, the enzyme is active if histidine is protonated and the carboxyl ionized. With fumarate as a substrate, the V profile is again bell-shaped showing two p a s. 7.0 and 4.9, respectively this time, however, the temperature variation indicates that p a 7.0 corresponds to dissociation of a carboxyl and p/iTa 4.9 to dissociation of a histidine side chain. Thus, from the fumarate side of reaction, the enzyme is active if carboxyl is protonated and histidine is not. 

Based on these results, Goldberg et al. (2006) suggested two possibilities to explain these findings. The first is that in R. oryzae the cytosolic fumarase is kinetically different from the mitochondrial isoenzyme, due to distinct posttranslational modifications, or to specific conditions in the two compartments. The second possibility is that R. oryzae harbors two genes encoding two different fumarases, one in mitochondria, which catalyzes the conversion of fumaric to L-malic acid, and a cytosolic enzyme, which catalyzes the conversion of L-malic to fumaric acid. Upon transfer into medium C, a fumarase with unique characteristics is induced. L-malic acid s conversion to fumaric acid is enhanced by the induced fumarase and when the concentration of fumaric acid in the cell exceeds 2 mM, the reverse reaction to L-malic acid is fully inhibited. Thus, this property of the unique fumarase, whose existence was then only hypothesized, can ensure that fumaric acid is accumulated. 

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