BIOL/BIOC 393 L03
Dr. Judit Moldovan
Submitted: Nov. 22nd, 2010
By: Jackie Minnick (Partners Amanda Verwoerd & Kersti Ojamaa) Study of stereospecificity in mushroom tyrosinase and the inhibiting effects of thiourea, cinnamic acid and benzoic acid.
This paper reports experiments on the stereospecificity observed in the monophenolase and diphenolase activities of mushroom tyrosinase and the inhibiting effects of thiourea, cinnamic acid and benzoic acid. The enantiomer L-DOPA and D-DOPA were assayed. The Vmax and Km values obtained for each were different. Thus mushroom tyrosinase showed stereospecificity in its higher affinity towards L-DOPA than D-DOPA. The same Km values were obtained for cinnamic acid and benzoic acid as compared to the uninhibited L-DOPA. However, the Vmax values were different. Therefore, both cinnamic acid and benzoic acid exhibit non-competitive inhibition for tyrosinase. Thiourea showed different results for Vmax and Km, still indicating that thiourea is a non-competitive inhibitor of tyrosinase. It was found that cinnamic acid is the strongest inhibitor of the three.
Tyrosinase, also commonly called polyphenol oxidase, catalyses two reactions: ortho-hydroxylation of monophenols and aerobic oxidation of ortho-diphenols. Equations 1 and 2:
Monohydroxyphenol + O2 + AH2 → o-dihydroxyphenol + H2O +A(1)
o-dihydroxyphenol + ½ O2 → o-quinone + H2O(2)
Reaction 1 and 2 are referred to as the monohydroxyphenolase and o-dihydroxyphenolase activities of tyrosinase, respectively (Andrawis & Kahn, 1986).
Tyrosinase is a copper containing enzyme found in many plant and animal cells. It plays a central role in the enzymatic browning of some fruits and vegetables, as well as such processes as vertebrate pigmentation. The reaction is localized in pigment cells and produces the melanin substances that are seen as color to skin, hair, and eyes. Figure 1 shows one of the steps in this process (Espin et al, 1998).
Figure 1. Overall reaction catalyzed by tyrosinase with the substrate L-DOPA, a colorless reactant, to result in the formation of the dopachrome product with orange color.
Tyrosinase has various natural sources. Mushroom tyrosinase is a protein, with two atoms of copper associated with the active enzyme. Two types of substrate binding sites exist in the enzyme, one type for the phenolic substrate and one type for the dioxygen molecule (Espin et al, 1998). Different tyrosinases from different sources have similar structural and functional characteristics (Robb, 1984).
Some enzymes will bind to a variety of substrates, if the structure of the substrate is similar, where as others are extremely specific, and will distinguish between D and L isomers. Several monophenols and o-diphenols are chiral tyrosinase substrates such as 3,4-Dihydroxy-L-Phenylalanine (commonly called L-DOPA) which is a natural substrate for mushroom tyrosinase. Its enantiomer, is commonly called D-DOPA.
Stereospecificity in several tyrosinases on L and D DOPA have been reported (Winder & Harris, 1991; Sonesson et al, 1995). Espin et al. (1998) reported the same Vmax values for each series of isomers at the same enzyme concentrations while the Km values were different. The sequence of Km values reported indicated D-isomers had lower affinity than L-isomers.
Not only has it been found that tyrosinase is stereospecific, but tyrosinase can be inhibited by aromatic aldehydes and aromatic acids (Shi et al, 2005). Cinnamic acid has been reported to inhibit the activity of mushroom tyrosinase for the oxidation of L-DOPA (Shi et al, 2005). Liu, Huang, and Chen (2003) reported that benzoic acid also had inhibitory effects on the diphenolase activity of mushroom tyrosinase. Moreover, they reported the benzaldehyde family of compounds showed...