Portal de Periódicos da CAPES

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês

10.1074/jbc.273.48.31661

1083-351X

Sonia Beeckmans, Edilbert Van Driessche,

Glycosylation and Glycoproteins Research

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substratesl-malate and fumarate, the active sites (sites A) also bind their analogs d-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogsd-malate and oxaloacetate, as well asl-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (K d being in the millimolar range) is 1–2 orders of magnitude lower than the affinity of sites A (of which K d is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (K d = 0.6 to 2.2 μm, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B). A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substratesl-malate and fumarate, the active sites (sites A) also bind their analogs d-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogsd-malate and oxaloacetate, as well asl-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (K d being in the millimolar range) is 1–2 orders of magnitude lower than the affinity of sites A (of which K d is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (K d = 0.6 to 2.2 μm, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B). l-1-tosylamido-2-phenylethyl chloromethyl ketone polyacrylamide gel electrophoresis. Fumarase (fumarate hydratase, EC 4.2.1.2) catalyzes the reversible, stereospecific addition of water to fumarate to forml-malate (1Hill R.L. Teipel J.W. Boyer P.D. The Enzymes. 3rd Ed. 5. Academic Press, New York1971: 539-571Google Scholar). Being an enzyme of the citric acid cycle, it serves both catabolic and anabolic purposes and, as such, it is found within all living organisms. In eukaryotes, both mitochondrial fumarase (which is involved in the citric acid cycle) and the cytoplasmic enzyme are encoded by the same gene (2Suzuki T. Sato M. Yoshida T. Tuboi S. J. Biol. Chem. 1989; 264: 2581-2586Abstract Full Text PDF PubMed Google Scholar, 3Suzuki T. Yoshida T. Tuboi S. Eur. J. Biochem. 1992; 207: 767-772Crossref PubMed Scopus (33) Google Scholar, 4Wu M. Tzagoloff A. J. Biol. Chem. 1987; 262: 12275-12282Abstract Full Text PDF PubMed Google Scholar). These fumarase molecules are tetramers consisting of identical subunits of 50 kDa each, and their activity does not depend upon the presence of metal cations (1Hill R.L. Teipel J.W. Boyer P.D. The Enzymes. 3rd Ed. 5. Academic Press, New York1971: 539-571Google Scholar). In prokaryotes on the other hand, there are two distinct classes of fumarase molecules: class I fumarases are heat-labile and Fe2+-dependent, dimeric enzymes with subunits of 60 kDa each, having no obvious sequence homology to the eukaryotic enzymes, whereas class II fumarases are heat-stable, Fe2+-independent tetrameric enzymes with subunits of 50 kDa and showing extensive homology to the eukaryotic fumarases. Recently the three-dimensional structure of Escherichia coli class II fumarase has been unraveled (5Weaver T.M. Levitt D.G. Donnelly M.I. Wilkens Stevens P.P. Banaszak L.J. Nat. Struct. Biol. 1995; 2: 654-662Crossref PubMed Scopus (116) Google Scholar, 6Weaver T. Banaszak L. Biochemistry. 1996; 35: 13955-13965Crossref PubMed Scopus (87) Google Scholar, 7Weaver T. Lees M. Banaszak L. Protein Sci. 1997; 6: 834-842Crossref PubMed Scopus (46) Google Scholar). According to crystallographic data, each subunit is composed of three domains, of which the central one (D2) forms a 5-helix bundle. The association of the D2 domains was shown to result in tetramer formation, domains D1 and D3 capping at opposite ends the central core composed by the 20 roughly parallel α-helices. From studies with nitrocarbanion substrate analogs on the one hand (8Porter D.J.T. Bright H.J. J. Biol. Chem. 1980; 255: 4772-4780Abstract Full Text PDF PubMed Google Scholar), and investigations of isotope effects on the other hand (9Blanchard J.S. Cleland W.W. Biochemistry. 1980; 19: 4506-4513Crossref PubMed Scopus (87) Google Scholar), it was unequivocally concluded that the mechanism of the fumarase reaction initially involves the formation of a carbanion. Proton removal froml-malate thus is the first chemical step of the dehydration reaction. The abstracted proton is stabilized onto the enzyme and is only released after fumarate (10Hansen J.N. Dinovo E.C. Boyer P.D. J. Biol. Chem. 1969; 244: 6270-6279Abstract Full Text PDF PubMed Google Scholar). The kinetics of the reaction catalyzed by pig heart fumarase have been studied in the past by several authors and have been summarized by Alberty (11Alberty R.A. Boyer P.D. The Enzymes. 2nd Ed. 5. Academic Press, New York1959: 531-544Google Scholar) and Hill and Teipel (1Hill R.L. Teipel J.W. Boyer P.D. The Enzymes. 3rd Ed. 5. Academic Press, New York1971: 539-571Google Scholar). These investigations showed that, whereas at low substrate concentrations fumarase exhibits Michaelis-Menten kinetics giving classic saturation curves in thev versus [S] plots, at substrate concentrations higher than 5 × K m substrate activation occurs, and at substrate concentrations above 0.1 minhibition is observed. The latter is explained by the binding of two substrate molecules at the same time into the enzyme's active site, thereby forming non-efficient enzyme-substrate complexes. Several mechanisms can be put forward to explain substrate activation. (a) The enzyme preparation might either contain two different fumarase species or two non-convertible conformational forms of a single species, each enzyme form obeying classical Michaelis-Menten kinetics and having its own set of K m and V max values. No indications for heterogeneity can, however, be detected through electrophoretic or physicochemical analyses (12Beeckmans S. Kanarek L. Int. J. Biochem. 1982; 14: 453-460Crossref PubMed Scopus (10) Google Scholar, 13Beeckmans S. Kanarek L. Peeters H. Protides of the Biological Fluids. 32. Pergamon Press, New York1984: 1013-1016Google Scholar). Moreover, the existence of distinct but stable conformers is highly improbable since denaturation as well as enzyme modification processes progress through simple kinetics (see e.g. Refs. 14Hill R.L. Kanarek L. Brookhaven Symp. Biol. 1964; 17: 80-97PubMed Google Scholar and 15Beeckmans S. Kanarek L. Biochim. Biophys. Acta. 1983; 743: 370-378Crossref PubMed Scopus (5) Google Scholar). (b) Another mechanism is that of negative cooperativity. According to this model, binding of the first molecule of substrate into one of the four, initially similar binding sites decreases the affinity for substrate molecules of the other sites on the enzyme. This hypothesis is ruled out since Teipel and Hill (16Teipel J.W. Hill R.L. J. Biol. Chem. 1968; 243: 5679-5683Abstract Full Text PDF PubMed Google Scholar) found linear Scatchard plots from equilibrium dialysis experiments. (c) Still another mechanism explaining the activation implies the existence of additional substrate-binding sites, other than and spatially separated from the active sites. The affinity of these supplementary substrate-binding sites may be supposed to be lower, but once they are occupied, the catalytic rate at the level of the active sites is increased due to (slight) conformational changes in the enzyme. Alternatively, the second substrate-binding sites may be potential catalytic sites as well. A number of anions, especially phosphate, have also been shown to affect the activity of pig heart fumarase (1Hill R.L. Teipel J.W. Boyer P.D. The Enzymes. 3rd Ed. 5. Academic Press, New York1971: 539-571Google Scholar, 11Alberty R.A. Boyer P.D. The Enzymes. 2nd Ed. 5. Academic Press, New York1959: 531-544Google Scholar, 17Andersen B. Biochem. J. 1980; 189: 653-654Crossref PubMed Scopus (4) Google Scholar, 18Wharton C.W. Szawelski R.J. Biochem. J. 1982; 203: 351-360Crossref PubMed Scopus (34) Google Scholar, 19Hasinoff B.B. Davey J.P. Biochem. J. 1986; 235: 891-893Crossref PubMed Scopus (4) Google Scholar). They can be supposed to bind into the substrate-binding sites as well, thereby exerting an effect on the enzyme which is comparable to the effect induced by the substrates themselves. (d) However, more recently Rose and co-workers (20Rose I.A. Warms J.V.B. Kuo D.J. Biochemistry. 1992; 31: 9993-9999Crossref PubMed Scopus (25) Google Scholar, 21Rose I.A. Warms J.V.B. Yuan R.G. Biochemistry. 1993; 32: 8504-8511Crossref PubMed Scopus (22) Google Scholar, 22Rose I.A. Biochemistry. 1997; 36: 12346-12354Crossref PubMed Scopus (23) Google Scholar) suggested that activation of fumarase at high concentrations of l-malate or fumarate can be explained without assuming additional substrate-binding sites. According to their investigations with pig heart (20Rose I.A. Warms J.V.B. Kuo D.J. Biochemistry. 1992; 31: 9993-9999Crossref PubMed Scopus (25) Google Scholar, 21Rose I.A. Warms J.V.B. Yuan R.G. Biochemistry. 1993; 32: 8504-8511Crossref PubMed Scopus (22) Google Scholar) as well as with a yeast fumarase (22Rose I.A. Biochemistry. 1997; 36: 12346-12354Crossref PubMed Scopus (23) Google Scholar), the interconversion of substrate- and product-free so-called "isoforms" of the enzyme might be a rate-limiting step during catalysis. It was claimed that activation of fumarase at substrate concentrations above 5 ×K m can be explained solely by such a recycling of free enzyme molecules through various conformational states differing in substrate specificity and catalytic activity. In their model, substrate specificity of the isoforms will depend upon the conformation of the active sites, as well as upon their state of protonation. It was further shown with yeast fumarase (22Rose I.A. Biochemistry. 1997; 36: 12346-12354Crossref PubMed Scopus (23) Google Scholar) that this recycling of the enzyme involves at least two proton transfers and a conformational change. Depending upon the anions present in the solution, whether simple anions (e.g. Cl−, Pi, etc.) or substrate analogs, these events, together with the release of l-malate, will be rate-determining. Recent crystallographic studies on the prokaryotic E. colifumarase (class II) revealed the presence of two distinct binding sites per monomer for inhibitors (5Weaver T.M. Levitt D.G. Donnelly M.I. Wilkens Stevens P.P. Banaszak L.J. Nat. Struct. Biol. 1995; 2: 654-662Crossref PubMed Scopus (116) Google Scholar, 6Weaver T. Banaszak L. Biochemistry. 1996; 35: 13955-13965Crossref PubMed Scopus (87) Google Scholar, 7Weaver T. Lees M. Banaszak L. Protein Sci. 1997; 6: 834-842Crossref PubMed Scopus (46) Google Scholar). It was concluded from these studies that the active site (site A) comprises amino acid residues belonging to three different subunits. Residues Thr-100, Ser-139, Ser-140, and Asn-141 from the b-subunit, Thr-187, His-188 from the d-subunit, and Lys-324, Asn-326 from the c-subunit were supposed to form direct hydrogen bonds with substrates and inhibitors (5Weaver T.M. Levitt D.G. Donnelly M.I. Wilkens Stevens P.P. Banaszak L.J. Nat. Struct. Biol. 1995; 2: 654-662Crossref PubMed Scopus (116) Google Scholar, 6Weaver T. Banaszak L. Biochemistry. 1996; 35: 13955-13965Crossref PubMed Scopus (87) Google Scholar). Moreover a highly coordinated buried water molecule was found at the A site and supposed to be associated to residues Ser-98, Thr-100, Asn-141 from the b-subunit and His-188 from the d-subunit (6Weaver T. Banaszak L. Biochemistry. 1996; 35: 13955-13965Crossref PubMed Scopus (87) Google Scholar, 7Weaver T. Lees M. Banaszak L. Protein Sci. 1997; 6: 834-842Crossref PubMed Scopus (46) Google Scholar). An additional binding site (site B) is supposed to be nearby and is built up of residues belonging to one single subunit (the b-subunit), i.e.Arg-126, His-129, Asn-131, and Asp-132 (5Weaver T.M. Levitt D.G. Donnelly M.I. Wilkens Stevens P.P. Banaszak L.J. Nat. Struct. Biol. 1995; 2: 654-662Crossref PubMed Scopus (116) Google Scholar). Conclusive evidence that site A is indeed the enzyme's catalytic site was provided by site-directed mutagenesis experiments (7Weaver T. Lees M. Banaszak L. Protein Sci. 1997; 6: 834-842Crossref PubMed Scopus (46) Google Scholar). Indeed, the mutation H188N in the A-site resulted in an enzyme showing largely reduced specific activity, whereas the mutation H129N in the B-site was essentially without effect. In view of the above presented arguments, it seemed of interest to investigate substrate binding with the most well known eukaryotic fumarase from pig heart tissue. In this article we present evidence that this fumarase also possesses two kinds of sites into which they specifically bind substrates and substrate analogs. We describe further kinetic studies as well as inactivation/modification experiments extending our knowledge about these substrate-binding sites, and unraveling the inter-relationship between both types of sites. Moreover the dissociation constants for both types of binding sites have been determined for a whole series of substrate analogs. Fumarase substrates and inhibitors were proanalysis grade and purchased from Sigma, as well as benzylbromide and the proteases trypsin (TPCK1-treated) and subtilisin. All benzenecarboxylic acids were from Aldrich; pyromellitic acid was recrystallized from distilled water. Tetranitromethane was from Fluka AG. Solutions of urea were deionized immediately before use by passing them through a column of Amberlite MB-3 resin (Rohm and Haas Co.), and the concentration was estimated from the refractive index of the deionized solutions (23Fasman G.D. Methods Enzymol. 1963; 6: 928-957Crossref Scopus (93) Google Scholar). Acrylamide and N,N′-methylene bisacrylamide were obtained from Fluka AG; they were recrystallized, respectively, from acetone and chloroform. Fumarase was purified to homogeneity from pig heart tissue and stored in the presence of 55% saturated ammonium sulfate as described by Beeckmans and Kanarek (24Beeckmans S. Kanarek L. Eur. J. Biochem. 1977; 78: 437-444Crossref PubMed Scopus (41) Google Scholar). Enzyme solutions were prepared by dialyzing an appropriate amount of ammonium sulfate suspension against 10 mm potassium phosphate buffer, pH 8.0, until the crystals were dissolved, and then against the buffer of choice. Routinely, fumarase activity is measured spectrophotometrically withl-malate as substrate (50 mml-malate in 50 mm potassium phosphate buffer, pH 7.9) as described before (12Beeckmans S. Kanarek L. Int. J. Biochem. 1982; 14: 453-460Crossref PubMed Scopus (10) Google Scholar). One unit of activity is the amount of enzyme which catalyzes the formation of 1 μmol of fumarate per min at 25 °C; measurements are performed at 250 nm where εfumarate = 1,450 m−1cm−1. The specific activity of the fumarase used in this study was 550 units/mg. For kinetic analyses, substrate concentrations between 10 μm and 50 mm were used either in 10 mm Tris acetate buffer or in potassium phosphate buffer (1–10 mm) at different values of pH. When fumarate consumption was analyzed, the activity was assayed at 285 nm, where εfumarate = 180 m−1cm−1. Analysis of the results was performed using the program "Leonora," as described by Cornish-Bowden (25Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). With this program, kinetic measurements were fitted to the classical Michaelis-Menten equation by the method of least squares using dynamic weights (i.e.Leonora was allowed to select by itself the best weighting scheme from internal evidence in the experimental data obtained). UV-absorption spectra and enzyme activity measurements were determined with a double-beam Shimadzu 210 UV apparatus (Bausch and Lomb). Circular dichroism spectroscopy was performed on a Jasco 750 spectropolarimeter. The mean residue ellipticity [θλ] was calculated from the equation [θλ] = (MRW/100) × (θλ/c.d), where the mean residual weight (MRW) is 110, c is the enzyme concentration in g/ml, and d is the path length in dm. K d values for several substrates and inhibitors were determined from their protection against fumarase inactivation brought about either by urea or by chemical modification or proteolytic degradation. In all cases, first-order kinetics of inactivation are observed, and (log % activity) is plotted against time. As was described earlier by Beeckmans and Kanarek (15Beeckmans S. Kanarek L. Biochim. Biophys. Acta. 1983; 743: 370-378Crossref PubMed Scopus (5) Google Scholar), the dissociation constant of an inhibitor I can then be calculated from:K d = (E/EI) × [I], in which (E/EI) is determined from the equations:k o (E) + k f(EI) = k and (E) + (EI) = 1, which is based upon the fact that free enzyme, E, and fully occupied enzyme, EI, are inactivated at different rates, respectively, k o and k f;k is the rate constant at an intermediate inhibitor concentration [I]. The kinetic constants are calculated from:k·t½ = 0.693, wheret½ is the half-life time of the inactivation. When K d is determined from circular dichroism data, the following equation is used: (E/EI) = ([θ] − [θf])/([θo] − [θ]), where [θo] and [θf] are, respectively, the mean residue ellipticity, at a certain wavelength, of the free enzymeE and fully occupied enzyme EI, and [θ] is the ellipticity at an intermediate concentration [I]. All these studies were performed in 10 mm Tris acetate buffer in order to avoid any influence of anions (like e.g.phosphate), binding into specific sites on the enzyme. When protection by substrates was investigated, the enzyme was incubated during at least 30 min in the presence of the required amount of fumaric acid prior to denaturation in order to allow the equilibrium (malate/fumarate) to be established. All chemical modification reactions and proteolytic degradations were performed at 25 °C using 10 mm Tris acetate as buffer. Fumarase tyrosyl residues were modified at pH 8.0 by adding an appropriate amount of a stock solution of tetranitromethane (1 m reagent in absolute ethanol) to reach a final concentration of 2.5 mm (15Beeckmans S. Kanarek L. Biochim. Biophys. Acta. 1983; 743: 370-378Crossref PubMed Scopus (5) Google Scholar). Methionyl residues were modified at pH 6.8 by adding an appropriate amount of a stock solution of benzylbromide (2 m reagent in methanol) to reach a final concentration of 20 mm (26Rogers G.A. Shaltiel N. Boyer P.D. J. Biol. Chem. 1976; 251: 5711-5717Abstract Full Text PDF PubMed Google Scholar). Since benzylbromide is only slightly soluble in water, each fumarase solution was continuously stirred during the modification reaction. For studies of limited proteolysis, either trypsin at pH 8.0 or subtilisin at pH 7.3 were used. For trypsinolysis, a stock solution was made of 5 mg of trypsin (TPCK-treated) in 1 ml of 1 mm HCl. A ratio 1:5 (w/w) of trypsin:fumarase was used for digestion. For subtilisinolysis, a stock solution was made of 10 mg of subtilisin in 10 ml of 10 mm acetic acid and 10 mmCaCl2 at pH 5.2. A ratio of 1:80 (w/w) of subtilisin:fumarase was used for digestion. The inactivation during proteolysis was studied by immediately assaying the remaining activity. In order to perform also SDS-PAGE analysis, the degradation was stopped at different times by adding to each 50 μl of fumarase sample (containing 0.5 mg of enzyme per ml), 5 μl of a freshly prepared phenylmethylsulfonyl fluoride stock solution, consisting of 40 mm reagent in absolute ethanol. All samples were kept frozen until electrophoresis. SDS-PAGE was performed according to Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212859) Google Scholar) using 10% polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue. N-terminal sequence analysis of partially proteolyzed fumarase was performed by classical Edman degradation with a Beckman 890C sequencer, and the amino acid derivatives were analyzed as described (12Beeckmans S. Kanarek L. Int. J. Biochem. 1982; 14: 453-460Crossref PubMed Scopus (10) Google Scholar). In the absence of phosphate ions, fumarase kinetics have an anomalous character in the whole pH range considered (6.5–8.5). Eadie-Hofstee plots show two straight lines (see Fig. 1 as an example). At low substrate concentrations the calculated K m1 value is a real Michaelis constant which, moreover, corresponds very well with the dissociation constant K d determined from equilibrium dialysis (16Teipel J.W. Hill R.L. J. Biol. Chem. 1968; 243: 5679-5683Abstract Full Text PDF PubMed Google Scholar). At higher substrate concentrations, we can calculate a K½ value, i.e. the substrate concentration where the velocity of the enzymatic reaction is half-way between the first and the second V max(Fig. 1, inset). This K½ value represents the affinity for substrate of the proposed second substrate-binding sites; it clearly differs from theK m2 value, which can be calculated directly from the experimental results obtained at high substrate concentrations, but not taking into account the experimental points at low substrate concentrations. As can be seen from Table I, part A, values of K m1, K½, and K m2 depend upon pH in a different way. The values for both K m and K½ are found to fulfill the Haldane relationship within experimental error: K eq = 4.4 = ([M]/[F])eq = (V mF ×K m)/(V mM ×K F), where K m and K F are K m1,K½, or K m2 for malate, respectively, fumarate, and VmM, VmFthe corresponding V max for malate, respectively, fumarate determined in the same substrate concentration range.Table IKinetic constants for the fumarase catalyzed reaction at different values of pHA: in 10 mm Tris acetate bufferpHSite ASite BK m1MK m1FK m1,appK½MK½FK½,appK m2MK m2FK m2,appμmmm6.512.9 ± 1.16.2 ± 0.310.7 ± 0.46.3 ± 1.54.6 ± 0.75.9 ± 0.51.28 ± 0.310.90 ± 0.161.19 ± 0.106.814.2 ± 0.74.9 ± 0.510.5 ± 0.38.4 ± 1.03.2 ± 0.56.5 ± 0.42.20 ± 0.270.84 ± 0.151.69 ± 0.117.016.3 ± 0.44.8 ± 0.511.3 ± 0.39.1 ± 0.92.7 ± 0.56.3 ± 0.42.62 ± 0.290.80 ± 0.161.84 ± 0.137.324.2 ± 0.55.2 ± 0.314.4 ± 0.210.0 ± 0.52.1 ± 0.25.9 ± 0.23.32 ± 0.190.69 ± 0.091.94 ± 0.087.539.2 ± 0.45.7 ± 0.318.8 ± 0.210.5 ± 0.51.5 ± 0.35.0 ± 0.24.01 ± 0.180.52 ± 0.181.79 ± 0.148.079.6 ± 0.86.2 ± 0.424.9 ± 0.410.0 ± 0.80.7 ± 0.22.9 ± 0.23.80 ± 0.310.30 ± 0.121.20 ± 0.128.5160.0 ± 0.98.8 ± 0.638.3 ± 0.55.8 ± 0.80.3 ± 0.21.3 ± 0.21.93 ± 0.280.10 ± 0.080.44 ± 0.17B: influence of phosphate ions and l-aspartateBuffer usedpHK m1MV m1MK½MKm2MVm2Mμmmm10 mm tris acetate8.079.6 ± 0.845.6 ± 0.210.0 ± 0.83.80 ± 0.3183.6 ± 1.51 mmpotassium phosphate8.080.1 ± 0.745.8 ± 0.110.1 ± 0.63.82 ± 0.2484.6 ± 1.710 mm potassium phosphate8.01.70 ± 0.0893.2 ± 1.8100 mm potassium phosphate8.07.72 ± 0.28102.2 ± 2.210 mm tris acetate7.324.2 ± 0.531.2 ± 0.310.0 ± 0.53.33 ± 0.2758.2 ± 2.310 mm tris acetate + 100 mml-aspartate7.30.67 ± 0.1264.8 ± 1.9The following symbols are used. K m1M and K m1F are Michaelis constants for, respectively, l-malate and fumarate, determined at low substrate concentrations (i.e. up to 1 mm) using the program Leonora (25Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). K m2M and K m2F are Michaelis constants for, respectively, l-malate and fumarate at high substrate concentrations (i.e. above 5 mm), but without taking into account the experimental points at low substrate concentrations; they were determined using the same computer program.K½M and K½Fare substrate (respectively, malate and fumarate) concentrations where the velocity of the reaction is half-way between the first and the second maximal initial velocities V max.K m1,app, K m2,app, and K½,app are the apparent values for the equilibrium mixture of l-malate and fumarate (calculated as described under "Materials and Methods"). V m1Mand V m2M are initial velocities, expressed relative to v = 100 in standard conditions (i.e. 50 mml-malate + 50 mm potassium phosphate at pH 7.9); they were again determined using the Leonora program (25Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). Open table in a new tab The following symbols are used. K m1M and K m1F are Michaelis constants for, respectively, l-malate and fumarate, determined at low substrate concentrations (i.e. up to 1 mm) using the program Leonora (25Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). K m2M and K m2F are Michaelis constants for, respectively, l-malate and fumarate at high substrate concentrations (i.e. above 5 mm), but without taking into account the experimental points at low substrate concentrations; they were determined using the same computer program.K½M and K½Fare substrate (respectively, malate and fumarate) concentrations where the velocity of the reaction is half-way between the first and the second maximal initial velocities V max.K m1,app, K m2,app, and K½,app are the apparent values for the equilibrium mixture of l-malate and fumarate (calculated as described under "Materials and Methods"). V m1Mand V m2M are initial velocities, expressed relative to v = 100 in standard conditions (i.e. 50 mml-malate + 50 mm potassium phosphate at pH 7.9); they were again determined using the Leonora program (25Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1995Google Scholar). Based on the results obtained, an estimation can be made, at different pH values, of the apparent valuesK m1,app and K½,app for the equilibrium mixtures ofl-malate and fumarate (Table I, part A) by using the equations (16Teipel J.W. Hill R.L. J. Biol. Chem. 1968; 243: 5679-5683Abstract Full Text PDF PubMed Google Scholar), K m1,app = 5.4/{(1/K m1F) + (4.4/K m1M)} and K½,app = 5.4/{(1/K½F) + (4.4/K½M)}. From a number of ligands used in this study, their effect on fumarase kinetics was investigated.d-Malate (K i = 50 μm at pH 7.3) and oxaloacetate (K i = 8.5 μm at pH 7.3) are considered (16Teipel J.W. Hill R.L. J. Biol. Chem. 1968; 243: 5679-5683Abstract Full Text PDF PubMed Google Scholar) to be structural analogues of, respectively, l-malate (K m1M= 24 μm) and fumarate (K m1F = 5.2 μm). Also glycine is a competitive inhibitor of fumarase and we determined its inhibition constant to be K i = 4 mm(results not shown; measurements performed in 10 mm Tris acetate buffer, pH 7.3, with l-malate concentrations below 1 mm). Moreover, several benzenecarboxylic acids were found to be competitive inhibitors. Their inhibition constants were determined in 10 mm Tris acetate buffer at pH 7.3 and are given in Table II. Especially pyromellitic acid, which can be considered to be a "duplicated" analog of fumarate, appears to be an excellent inhibitor and was used before as affinity ligand for the purification of fumarases from several organisms (12Beeckmans S. Kanarek L. Int. J. Biochem. 1982; 14: 453-460Crossref PubMed Scopus (10) Google Scholar, 24Beeckmans S. Kanarek L. Eur. J. Biochem. 1977; 78: 437-444Crossref PubMed Scopus (41) Google Scholar, 28Weaver T.M. Levitt D.G. Banaszak L.J. J. Mol. Biol. 1993; 231: 141-144Crossref PubMed Scopus (16) Google Scholar). Its K i of 0.6 μm (at pH 7.3) is 1–2 orders of magnitude lower than theK m1 for the natural substrates. However, the presence of an additional carboxyl group appreciably interferes with bind