Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.).

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.

Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies.

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.

Mushroom tyrosinase: catalase activity, inhibition, and suicide inactivation.

Mushroom tyrosinase exhibits catalase activity with hydrogen peroxide (H(2)O(2)) as substrate. In the absence of a one-electron donor substrate, H(2)O(2) is able to act as both oxidizing and reducing substrate. The kinetic parameters V(max) and K(m) that characterize the reaction were determined from the initial rates of oxygen gas production (V(0)(O)()2) under anaerobic conditions. The reaction can start from either of the two enzyme species present under anaerobic conditions: met-tyrosinase (E(m)) and deoxy-tyrosinase (E(d)). Thus, a molecule of H(2)O(2) can reduce E(m) to E(d) via the formation of oxy-tyrosinase (E(ox)) (E(m) + H(2) <==> O(2) right harpoon over left harpoon E(ox)), E(ox) releases oxygen into the medium and is transformed into E(d), which upon binding another molecule of H(2)O(2) is oxidized to E(m). The effect of pH and the action of inhibitors have also been studied. Catalase activity is favored by increased pH, with an optimum at pH = 6.4. Inhibitors that are analogues of o-diphenol, binding to the active site coppers diaxially, do not inhibit catalase activity but do reduce diphenolase activity. However, chloride, which binds in the equatorial orientation to the protonated enzyme (E(m)H), inhibits both catalase and diphenolase activities. Suicide inactivation of the enzyme by H(2)O(2) has been demonstrated. A kinetic mechanism that is supported by the experimental results is presented and discussed.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism.

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.

Compound I formation in artichoke (Cynara scolymus L.) peroxidase is modulated by the equilibrium between pentacoordinated and 6-aquo hexacoordinated forms of the heme and by calcium ions.

Basic artichoke (Cynara scolymus L.) peroxidase (AKP-C), when purified from the plant, has an unusually intense and sharp Soret absorption peak. The resonance Raman spectrum [López-Molina, D., et al. (2003) J. Inorg. Biochem. 94, 243-254] suggested a mixture of pentacoordinate high-spin (5cHS) and 6-aquo hexacoordinate high-spin (6cHS) ferric heme species. The rate constant (k(1)) of compound I formation with hydrogen peroxide (H(2)O(2)) was also lower than expected. Further stopped-flow studies have shown this reaction to be biphasic: a nonsaturating fast phase and a slow phase with complex H(2)O(2) concentration dependence. Addition of calcium ions (Ca(2+)) changed the absorption spectrum, suggesting the formation of a fully 5cHS species with a k(1) more than 5 orders of magnitude greater than that in the absence of Ca(2+) using the chelator ethylenediaminetetraacetic acid. Ca(2+) titrations gave a dissociation constant for a single Ca(2+) of approximately 20 microM. The circular dichroism spectrum of AKP-C was not significantly altered by Ca(2+), indicating that any structural changes will be minor, but removal of Ca(2+) did suppress the alkaline transition between pH 10 and 11. A kinetic analysis of the reaction of Ca(2+)-free AKP-C with H(2)O(2) supports an equilibrium between a slow-reacting 6cHS form and a more rapidly reacting 5cHS species, the presence of which was confirmed in nonaqueous solution. AKP-C, as purified, is a mixture of Ca(2+)-bound 5cHS, 6-aquo 6cHS, and Ca(2+)-free 5cHS species. The possibility that Ca(2+) concentration could control peroxidase activity in the plant is discussed.

Mechanisms of compound I formation in heme peroxidases.

The formation of compound I is the first step in the reaction mechanism of plant heme peroxidases. This intermediate stores two oxidizing equivalents from hydrogen peroxide as an oxyferryl iron center and a radical, either on the porphyrin ring or on a tryptophan residue. Site-directed mutagenesis has proved to be a most useful tool for the identification of the intermediates involved and the resulting nature of the compound I formed. Although there is no doubt that an acid-base mechanism operates in heme peroxidase during the formation of compound I, the roles of several distal pocket residues are currently the subject of intensive research. It is now generally accepted that the conserved distal histidine in the active site of heme peroxidases is the acid-base catalyst that promotes the heterolytic cleavage of hydrogen peroxide. Other residues, such as the distal arginine and asparagine, participate in a range of roles assisting catalysis by the distal histidine. Recent advances in the elucidation of the mechanism at the molecular level are discussed. Another aspect related to the nature of compound I is the location of the radical center. Novel radical species have been detected in the reactions of ascorbate peroxidase, lignin peroxidase and several mutants of horseradish peroxidase. Detailed kinetic and spectroscopic studies of these radical species have provided important insights about the factors that control porphyrin-protein radical exchange. The wide range of data being obtained on compound I will lead to an understanding of its vital function in peroxidase catalysis and the physiological roles played by these enzymes.

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation.

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.

Mechanistic implications of variable stoichiometries of oxygen consumption during tyrosinase catalyzed oxidation of monophenols and o-diphenols.

The stoichiometry of oxygen consumption during tyrosinase-catalyzed oxidation of an o-diphenol (4-tert-butylcatechol, TBC) and a monophenol (4-tert-butylphenol, TBP) has been determined. At high [substrate]/[enzyme] ratios, in the case of o-diphenols, the stoichiometry of the enzyme-catalyzed reaction was always 1 O(2)/2 o-diphenols, although if the o-quinone product was unstable, the apparent stoichiometry could tend to 1 O(2)/1 o-diphenol due to regeneration of an o-diphenol in a side reaction. In the case of monophenols, the stoichiometry could be 1 O(2)/1 monophenol or 1.5 O(2)/1 monophenol depending if the o-quinone product was stable or unstable, respectively. However, at low [substrate]/[enzyme] ratios, the oxygen/substrate stoichiometry could, even in the case where stable products are formed, be lower than 1 O(2)/2 substrates for o-diphenols or higher than 1 O(2)/1 substrate for monophenols. These data supported the mechanism proposed by Rodríguez-López et al. [J. Biol. Chem. 267 (1992) 3801-3810], in which, during hydroxylation of monophenols, tyrosinase first transformed monophenol to o-diphenol and then either catalyzed a further oxidation to form o-quinone or released it into the reaction medium. In this second case, subsequent oxidation of the o-diphenol resulted in additional oxygen consumption.