Unification for the Expression of the Monophenolase and Diphenolase Activities of Tyrosinase (original) (raw)
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Spectrophotometric determination of monophenolase activity of tyrosinase isozymes
Archives of Biochemistry and Biophysics, 1971
Mushroom tyrosinase (EC 1.10.3.1, o-diphenol:oxygen oxidoreductase) was partially purified and the oxidation of p-cresol studied. The conditions were established for a spectrophotometric determination of monophenolase (cresolase) activity, based on measurements of initial velocity. Tyrosinase isozymes were separated by high-resolution column chromatography on Sephadex A-50 (DEAE), and distinguished by substrate speeificities. The ratio of classical manometric cresolase units (13) to spectrophotometric units was 3.85 for all isozymes.
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;
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.
Purification of tyrosinase from edible mushroom
Iranian Journal of Biotechnology, 2004
A simple preparative method was developed for purification of Tyrosinase from edible mushroom (Agaricus bispora). A homogenized extract of mushroom was first saturated by ammonium sulfate. The desired precipitate was mixed thoroughly with DEAE-Cellulose (DE-52) and washed out to produce melanin free precipitate. The obtained protein solution was dialyzed against running water for 4 hrs, then, concentrated and chromatographed on a DE-52 column. On the basis of the activities assay, the eluted fractions by 150 mM salt solution were selected for further purification. The collected fractions were pooled and chromatographed on a Sephadex G-200 column. Polyacrylamide gel electrophoresis (PAGE) of the purified tyrosinase produced a single band right beside the commercial sample obtained from Sigma Company at 128 kDa. The lyophilized form of the purified Tyrosinase had a purification degree of 104 and showed strong cresolase and catecholase activities when compared to a commerically availab...
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.
Mushroom Tyrosinase: Recent Prospects
Journal of Agricultural and Food Chemistry, 2003
Tyrosinase, also known as polyphenol oxidase, is a copper-containing enzyme, which is widely distributed in microorganisms, animals, and plants. Nowadays mushroom tyrosinase has become popular because it is readily available and useful in a number of applications. This work presents a study on the importance of tyrosinase, especially that derived from mushroom, and describes its biochemical character and inhibition and activation by the various chemicals obtained from natural and synthetic origins with its clinical and industrial importance in the recent prospects.
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.
Biomolecules
With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A−ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 μM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenol...
Comparison of the characteristics of fungal and plant tyrosinases
Journal of Biotechnology, 2007
Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein-and peptide-bound tyrosyl residues, resulting in the formation of inter-and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, ␣-casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di-or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein ␣-casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink ␣-casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink ␣-casein. In the reaction conditions studied, TrT showed the best ability to crosslink ␣-casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.