Suicide inactivation of tyrosinase in its action on tetrahydropterines (original) (raw)
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Indirect inactivation of tyrosinase in its action on tyrosine
2014
Under aerobic conditions, tyrosinase is inactivated by dopa as a result of suicide inactivation, and, under anaerobic conditions, as a result of irreversible inactivation. However, tyrosine protects the enzyme from being inactivated by dopa under anaerobic conditions. This paper describes how under aerobic conditions the enzyme acting on tyrosine is not directly inactivated but undergoes a process of indirect suicide inactivation provoked by reaction with the o-diphenol originated from the evolution of o-dopaquinone and accumulated in the reaction medium.
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...
Tetrahydrofolic Acid Is a Potent Suicide Substrate of Mushroom Tyrosinase
Journal of Agricultural and Food Chemistry, 2011
The coenzyme tetrahydrofolic acid is the most rapid suicide substrate of tyrosinase that has been characterized to date. A kinetic study of the suicide inactivation process provides the kinetic constants that characterize it: λ max , the maximum apparent inactivation constant; r, the partition ratio or the number of turnovers made by one enzyme molecule before inactivation; and k cat and K m , the catalytic and Michaelis constants, respectively. From these values, it is possible to establish the ratio λ max /K m , which represents the potency of the inactivation process. Besides acting as a suicide substrate of tyrosinase, tetrahydrofolic acid reduces o-quinones generated by the enzyme in its action on substrates, such as L-tyrosine and L-DOPA (o-dopaquinone), thus inhibiting enzymatic browning.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2012
We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines b o-aminophenols b o-diphenols, which agrees with the view that the transfer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle. As regards the apparent inactivation constant, it remains within the same order of magnitude, although slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-diamines b o-aminophenols b o-diphenols. The efficiency of the second nucleophilic attack of substrate on CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways. This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the number of turnovers that 1 mol of enzyme carries out before being inactivated, follows the order aromatic o-diamines b o-aminophenols b o-diphenols. As regards the Michaelis constants, that of the o-diamines is slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λ max /K m S , follows this order: o-diphenols > o-aminophenols > aromatic o-diamines.
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.
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 ).
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.
A Further Step in the Kinetic Characterisation of the Tyrosinase Enzymatic System
Journal of Mathematical Chemistry, 2006
Tyrosinase is a cuproprotein that hydroxylates monophenols to o-diphenols, which it then oxidises to o-quinones, using molecular oxygen. Based on kinetic studies of the steady state and measuring product formation during the action of the enzyme on o-diphenols, we determine the Michaelis constant and the maximum velocity, respectively. Similarly, we determine these kinetic constants for the enzyme acting on monophenols. From these constants obtained for a monophenol/o-diphenol pair, it is possible to calculate a new constant, the Michaelis constant of the enzyme for an o-diphenol acting on the corresponding monophenol, by means of an equation that relates the above-mentioned kinetic constants. Furthermore, it is also possible to establish the relation between the Michaelis constants for the oxygen in the presence of monophenol and in the presence of o-diphenol from the relation between the maximum velocities of the monophenol and o-diphenol experimentally determined by measuring aminochrome. From applying the equations described above to the kinetic data of the many tyrosinases described in the literature, we find that the Michaelis constant for the o-diphenol in the presence of monophenol is much lower than that obtained when the enzyme acts on o-diphenol alone. The Michaelis constant for oxygen in the presence of monophenol is also much lower than that obtained in the presence of its o-diphenol.
Indirect inactivation of tyrosinase in its action on 4- tert -butylphenol
Journal of Enzyme Inhibition and Medicinal Chemistry, 2013
Under aerobic conditions, tyrosinase is inactivated by dopa as a result of suicide inactivation, and, under anaerobic conditions, as a result of irreversible inactivation. However, tyrosine protects the enzyme from being inactivated by dopa under anaerobic conditions. This paper describes how under aerobic conditions the enzyme acting on tyrosine is not directly inactivated but undergoes a process of indirect suicide inactivation provoked by reaction with the o-diphenol originated from the evolution of o-dopaquinone and accumulated in the reaction medium.
European Journal of Biochemistry, 1987
The catalytic mechanism of tyrosine 3-monooxygenase (tyrosine hydroxylase, EC 1.14.1 6.2), isolated from the cytosolic fraction of bovine adrenal medulla, was studied by new techniques of product isolation and characterization. Using either (6R)-tetrahydrobiopterin, (6RS)-tetrahydroneopterin, or 6-methyl-tetrahydropterin, as the cofactors, three enzymatic oxidation products could be isolated and identified from the reaction mixture by high-performance liquid chromatography and rapid-scanning spectroscopy: (a) the 4a-hydroxy derivatives, (b) the quinonoid dihydropterins, and (c) the stable 7,8-dihydropterins.