Tetrahydrofolic Acid Is a Potent Suicide Substrate of Mushroom Tyrosinase (original) (raw)
Related papers
Substrate share in the suicide inactivation of mushroom tyrosinase
Biochimica et Biophysica Acta (BBA) - General Subjects, 2004
To address the real cause of the suicide inactivation of mushroom tyrosinase (MT), under in vitro conditions, cresolase and catecholase reactions of this enzyme were investigated in the presence of three different pairs of substrates, which had been selected for their structural specifications. It was showed that the cresolase activity is more vulnerable to the inactivation. Acetylation of the free tyrosyl residues of MT did not cure susceptibility of the cresolase activity, but clearly decreased the inactivation rate of MT in the presence of 4-[(4methylbenzo)azo]-1,2-benzenediol (MeBACat) as a catecholase substrate. Considering the results of the previous works and this research, some different possible reasons for the suicide inactivation of MT have been discussed. Accordingly, it was proposed that the interruption in the conformational changes in the tertiary and quaternary structures of MT, triggered by the substrate then mediated by the solvent molecules, might be the real reason for the suicide inactivation of the enzyme. However, minor causes like the toxic effect of the ortho-quinones on the protein body of the enzyme or the oxidation of some free tyrosyl residues on the surface of the enzyme by itself, which could boost the inactivation rate, should not be ignored.
Phenolic substrates and suicide inactivation of tyrosinase: kinetics and mechanism
Biochemical Journal, 2008
The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form Eox (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon...
Mushroom Tyrosinase: Catalase Activity, Inhibition, and Suicide Inactivation
Journal of Agricultural and Food Chemistry, 2005
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 h 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.
Journal of Agricultural and Food Chemistry, 2007
The inhibitory characteristics of two isoflavone metabolites, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′tetrahydroxyisoflavone, on mushroom tyrosinase were investigated. The two isoflavones were isolated from soygerm koji and inhibited both monophenolase and diphenolase activities of tyrosinase. Their inhibition type was demonstrated to be irreversible inhibition by preincubation and recovery experiments. By using HPLC analysis, it was found that mushroom tyrosinase could catalyze the two isoflavones. These results revealed that the two isoflavones belonged to suicide substrates of mushroom tyrosinase. The partition ratios between molecules of suicide substrate in the formation of product and in the inactivation of enzyme were determined to be 81.7 (5.9 and 35.5 (3.8 for 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone, respectively. From kinetic studies, maximal inactivation rate constants and Michaelis constants were 0.79 (0.08 and 1.01 (0.04 min-1 and 18.7 (2.31 and 7.81 (0.05 µM for 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone, respectively, when L-DOPA was used as the enzyme substrate. Structure analysis comparing the inactivating activity between the two isoflavones and their structure analogues showed that not only the 7,8-dihydroxyl groups but also the isoflavone skeleton of the two isoflavones played an important role in inactivating tyrosinase activity. The present study demonstrated that 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone are potent suicide substrates of mushroom tyrosinase.
International Journal of Biological Macromolecules, 2004
Modification (acetylation) of Tyr residues with N-acetylimidazole protects outstandingly mushroom tyrosinase (MT) from the suicide inactivation in the presence of its catecholic substrate, 4-[(4-methylbenzo) azo]-1,2-benzenediol. UV spectrophotometric experiments and differential scanning calorimetry (DSC) studies indicated a decrease in kinetic stability of the enzyme alongside with increase in its thermal stability as well as its stability against n-dodecyl trimethylammonium bromide as a denaturizing agent. Pace analysis resulted in standard Gibbs free energy values of 46.54 and 52.09 kJ/mol in the absence of denaturant for native and modified enzyme, respectively. Structural studies by circular dichroism (CD) spectrophotometry showed that modification did not have major impact on the secondary structure of MT; however, induced some changes in its tertiary structure. The near-UV CD results revealed that the modification had enhanced intramolecular van der Waals interactions in the enzyme structure, which was in coincidence with its thermodynamic stability.
Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase
European Journal of Biochemistry, 2000
This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V M max values for each monophenol was found. In the case of o-diphenols the same effect was observed but the size of the side-chain became very important. NMR studies on the monophenols justified the sequence of the V M max values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and V D max values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step k 33 is not the rate-determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack k 5 1 of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k 5 2 ) of the oxygen atom of the active site of oxytyrosinase on the C-3 of the monophenolic ring. However, the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position k 32 and to the size of the substituent side-chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed. Abbreviations: d 3 , chemical displacement value at C-3; d 4 , chemical displacement value at C-4; D, o-diphenol; 2 H 2 O, deuterium oxide (heavy water); [D] 0 , initial o-diphenol concentration; [D] ss , o-diphenol concentration in the steady-state; [D] 1 , o-diphenol concentration at the final assay time; DHPAA, 3,4-dihydroxyphenyl acetic acid; DHPPA, 3,4-dihydroxyphenyl propionic acid; DMF, N, N H -dimethylformamide; : max , molar absorptivity at l max ; : i , molar absorptivity at l i ; E d , deoxy-tyrosinase (reduced form of tyrosinase with Cu 1 -Cu 1 in the active site); E m , met-tyrosinase (with Cu 21 -Cu 21 in the active site); E o , oxy-tyrosinase with Cu 21 -O 2 22 -Cu 21 in the active site; 4HA, 4-hydroxyanisole; k D cat , catalytic constant of tyrosinase towards o-diphenols; k M cat , catalytic constant of tyrosinase towards monophenols; K 1 = k 21 k 1 ; K 2 = k 22 k 2 ; K 6 = k 26 k 6 ; K 4 = k 24 k 4 ; K D m , apparent Michaelis constant of tyrosinase towards o-diphenols; K M m , apparent Michaelis constant of tyrosinase towards monophenols; l max , wavelength at the maximum of absorbance; l i , wavelength at the isosbestic point; M, monophenol;
SURVEYING ALLOSTERIC COOPERATIVITY AND COOPERATIVE INHIBITION IN MUSHROOM TYROSINASE
Journal of Food Biochemistry, 2010
ABSTRACTIn view of the increasing importance of controlling tyrosinase activities, this paper addresses the true kinetics of both activities of MT. Eadie–Hofstee analysis of the kinetics results obtained from the direct spectrophotometric measurements of the cresolase activity in the presence of p-coumaric acid and MePAPh and catecholase activity in the presence of caffeic acid and MePACat showed deviation from linearity at lower and higher concentrations of the substrates. Comprehensive kinetics studies on both activities of MT resulted in typical saturation curves of the enzymes displaying mixed cooperativity. The analysis of the double-reciprocal plots and the slope analysis of the Hill plots of the kinetics data indicate negative cooperativity in both activities. However, it is more pronounced in the cresolase activity. Using the results of some inhibition studies on the cresolase activity, the obtained kinetics results have been explained and discussed in terms of allosteric cooperativity, cooperative inhibition and mixed-cooperativity.In view of the increasing importance of controlling tyrosinase activities, this paper addresses the true kinetics of both activities of MT. Eadie–Hofstee analysis of the kinetics results obtained from the direct spectrophotometric measurements of the cresolase activity in the presence of p-coumaric acid and MePAPh and catecholase activity in the presence of caffeic acid and MePACat showed deviation from linearity at lower and higher concentrations of the substrates. Comprehensive kinetics studies on both activities of MT resulted in typical saturation curves of the enzymes displaying mixed cooperativity. The analysis of the double-reciprocal plots and the slope analysis of the Hill plots of the kinetics data indicate negative cooperativity in both activities. However, it is more pronounced in the cresolase activity. Using the results of some inhibition studies on the cresolase activity, the obtained kinetics results have been explained and discussed in terms of allosteric cooperativity, cooperative inhibition and mixed-cooperativity.PRACTICAL APPLICATIONSTyrosinase is a widespread enzyme with great promising capabilities. The past decade research has just started to unfold some other important roles of tyrosinases. In spite of the outstanding progress of the tyrosinase science, there are still some important and immediate issues which have to be addressed. Considering the massive number of different formulations in the healthcare and cosmetic market which contain tyrosinase controlling substances, it seems that elucidation of true nature of the tyrosinase kinetics has become a necessity. Besides, tyrosinases are important subjects of many ongoing researches mainly due to their key role in the enzymatic browning phenomenon which affects the quality of fruits, vegetables and crop products.The outcome of this research can be quite meaningful to the current studies on controlling tyrosinase activities in medicine, nutrition, physiology and biotechnology.Tyrosinase is a widespread enzyme with great promising capabilities. The past decade research has just started to unfold some other important roles of tyrosinases. In spite of the outstanding progress of the tyrosinase science, there are still some important and immediate issues which have to be addressed. Considering the massive number of different formulations in the healthcare and cosmetic market which contain tyrosinase controlling substances, it seems that elucidation of true nature of the tyrosinase kinetics has become a necessity. Besides, tyrosinases are important subjects of many ongoing researches mainly due to their key role in the enzymatic browning phenomenon which affects the quality of fruits, vegetables and crop products.The outcome of this research can be quite meaningful to the current studies on controlling tyrosinase activities in medicine, nutrition, physiology and biotechnology.
Studies of the competing rates of catechol oxidation and suicide inactivation of tyrosinase
Arkivoc, 2010
Tyrosinase oxidation of catechols to ortho-quinones is accompanied by suicide inactivation of the enzyme. The rates of these competing processes vary and depend on the nature of ring substituents. For a series of 4-substituted catechols the relationships between structure and reaction rates have been examined using multiple regression. Significant but different structurerate relationships were found for each process. The oxidation rate (k 1 ) is greatest for short hydrophobic substituents; there is an optimum substituent hydrophobicity (π ~ 0.7) for the rate of inactivation (k 2 ).
Analytical Biochemistry, 2003
Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their k max (s) with the least interference of the intermediatesÕ or productsÕ absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented.
International Journal of Biochemistry & Cell Biology, 2002
The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and oxygen were derived for both, monophenolase and diphenolase activities of the enzyme. Thus, the Michaelis constants of tyrosinase towards the oxygen (K mO 2 ) are related with the respective catalytic constants for monphenols (k M cat ) and o-diphenols (k D cat ), as well as with the rate constant, k +8 . We recently determined the experimental value of the rate constant for the binding of oxygen to deoxytyrosinase (k +8 ) by stopped-flow assays. In this paper, we calculate theoretical values of K mO 2 from the experimental values of catalytic constants and k +8 towards several monophenols and o-diphenols. The reliability and the significance of the values of K mO 2 are discussed.