Mushroom, tyrosinase

Shiino M, WatanabeY, UmezawaK (2001) Synthesis of N-substituted N-nitroso-hydroxylamines as inhibitors of mushroom tyrosinase. Bioorg Med Chem 9(5) 1233-1240... 

Table 12.5 Product formation in biotransformations with whole cells of P. mendocina KRl expressing T4MO alone and in tandem with mushroom tyrosinase s...

In Experiment 5, you will study kinetics and inhibition with the enzyme mushroom tyrosinase. Use PubMed bibliographic searches to learn the following aspects of the enzyme ... 

The many tyrosinases in nature differ in cofactor requirement, metal ion content, substrate specificity, molecular weight, and oligomeric structure. The diverse properties of the tyrosinases will not be discussed instead, the properties of a single enzyme, mushroom tyrosinase, will be outlined. 

Several kinetic characteristics of mushroom tyrosinase will be examined in this experiment. A spectrophotometric assay of tyrosinase activity will be introduced and applied to the evaluation of substrate specificity, Ku of the natural substrate, 3,4-dihydroxyphenylalanine (L-dopa), and inhibition characteristics. 

Sodium phosphate buffer, 0.05 M, pH 7. 0 Mushroom tyrosinase, 100 units/mL... 

I. Behbahani, S. Miller, and D. O Keeffe, Microchem.J. 47,251-260 (1993). A Comparison of Mushroom Tyrosinase Dopaquinone and Dopachrome Assays. ... 

H. Duckworth and J. Coleman,/. Biol. Chem. 245, 1613-1625 (1970). Kinetic properties of mushroom tyrosinase. 

Sodium phosphate buffer, 0.05 M, pH 7. 0 Mushroom tyrosinase, 100 umts/mL 1 unit represents the amount of enzyme that causes an A280 change of 0.001 using tyrosine as substrate. Prepare solution in phosphate buffer A280 should be about 0.20. Store in ice bath dunng laboratory period. 

PV Ramsohoye, IA Kozlov. Isoprotein composition and cross-linking of thaumatins using mushroom tyrosinase and dimethyl suberimidate. Int J Food Sci Technol 26(3) 271-282, 1991. 

Pandey G, Muralikrishna C, Bhalerao UT (1989) Mushroom tyrosinase catalysed synthesis of coumestans, bebzofuranderivatives and related heterocyclic compounds. Tetrahedron 45 6867... 

Tyrosinase inhibitors prevent browning in foodbecause they inhibit the oxidation caused by the enzyme tyrosinase. Cuminaldehyde is identified as a potent mushroom tyrosinase monophenol monooxygenase inhibitor from cumin seeds, ft inhibits the oxidation of L-3,4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase with an ID50 of 7.7g/ml (0.05 mM). Its oxidized analogue, cumic acid (p-isopropylbenzoic acid), also inhibits this oxidation with an 1D50 of 43g/ml (0.26mM). These two inhibitors affect mushroom tyrosinase activity in different ways (Kubo and Kinst-Hori, 1998). 

Means within each protein concentration not followed by the same letter are significantly different at p <0.05. PPO (mushroom tyrosinase) was added at 0.100 change in OD yQ/min/gm diet as chlorogenic acid oxidase activity and chlorogenic acid at 3.5 umoles/gm wet weight. 

Mushroom tyrosinase has previously been used to convert tyrosine residues in chemically synthesized polyphenolic decapeptides to dopa residues (38), This enzyme also can convert dopa residues to quinones, but the enzymatic product can be maintained in the dopa form if reducing conditions are utilized. Using mushroom tyrosinase, we have converted at least 50% of the tyrosine residues to dopa and have evidence for quinone-lysine crosslinks in an oxidizing environment (T. Wei and R. Link, unpublished data). When these conditions are carefully controlled, we have observed adhesive properties for the recombinant polyphenolic protein. We are currently studying the parameters that can increase adhesivity and moisture resistance through better surface interactions and more extensive crosslinking. 

Tyrosinase catalyzes the oxidation of phenols. These enzymes are widespread in fungi, plants, and animals. Polyhydroxystyrene (PHS) is a phenol-containing polymer used as the excellent polymer matrix due to its good coating properties. Phenol moieties of PHS can be oxidized by tyrosinase. Mushroom tyrosinase was observed to catalyze the oxidation of 1-2% phenolic moieties of the synthetic polymer poly (4-hydroxystyrene) (PHS) (Shao et al., 1999) (Figure 4.5). 

The standard reaction mixture contained 0.3 fig of mushroom tyrosinase in 300 fiL of 0.05 M Mops buffer (pH 6.5) and 3.6 /tmol of 3,4-dihydroxybenzaldehyde dissolved in 300 fiL of the same buffer. The reaction mixture was incubated at 30°C, and aliquots were withdrawn at various times up to 10 minutes, with each added to an equal volume of ice-cold 0.2 M perchloric acid. After centrifugation, 2 to 5 fiL was analyzed by HPLC. For single time-point determinations, the reaction volume was reduced to 50 fiL. Product formation was linear with time to 10 minutes and with the amount of protein added. 

The assay was used to measure the activity of a commercial preparation of mushroom tyrosinase, and the activity in cell-free hemolymph from mosquitoes. 

Figure 9.54 Standard mushroom tyrosinase reaction that was stopped by the addition of an equal volume of 0.2 M perchloric acid containing 120 mAf DHBZ. After centrifugation, 2 fiL of resulting supernatant was injected. (From Li et at., 1990.)...

A two-electron oxidation of N-acetyltyrosine ethyl ester with mushroom tyrosinase, or with periodate, afforded the N-acetyIdopa ester 142, together with the (Z)-enamide 145 and the 6-acetoxydopa amide 146 (Fig. 40) (284). It is assumed that 145 originates from dopaquinone 143 via 144 by tautomerization. Michael addition of acetate to quinone 143 is believed to be the origin of 146. The formation of quinone methide 144 from dopa ester 142 by tyrosinase is reminiscent of the formation of iminochromes and quinone methides catalyzed by this enzyme in their formation from a-methyl dopa ester (285), and such reactions may well occur in mammalian systems. 

Tyrosinase (see Copper Proteins with Dinuclear Active Sites), a copper metalloenzyme with a very broad phylogenetic distribution, is responsible for the browning of fruits and mushrooms.Tyrosinase is a bifimctional phenol oxidase that is able to both hydroxylate monophenols like tyrosine (monooxygenase reaction, (equations)) and snbseqnently oxidize the diphenol product to the corresponding quinone (oxidase reaction, (equation 6)) at a single Type 3 binuclear copper active site. 

Figure 7. Absorption spectra of oxyhemocyanin (—) from Loligo pealei and the H2O2 derivative of mushroom tyrosinase (—). Redrawn from (23, 24, 25, 26) and (72).

Figure 9. ESR spectra near g = 4 of the nitric oxide derivatives of mushroom tyrosinase (A) and hemocyanins from Helix pomatio (B) and Cancer magister (C). T — 14°K, power = 200 mW (54).

Figure 10, Spectral changes during the oxidation of 4-nitrocate-chol by mushroom tyrosinase (A) and fungal laccase (B) (102)...

Figure 11. Lineweaver-Burk plots of the oxidation of pyrocatechol (A), 4-acetylcatechol (B), and 4-formylcatechol (C) by mushroom tyrosinase at several oxygen tensions (102)...

Figure 14. Lineweaver-Burk plot and Eadie plot (inset) of the oxidation of la-DOFA by mushroom tyrosinase...

---