Background
Tyrosinase is an enzyme that is found across a broad range of organisms including bacteria, plants, fungi, and animals. It plays an important role in the browning of fruits, vegetables, and mushrooms when they are bruised. In mammals, tyrosinase plays a key role in the process of melanogenesis which is responsible for skin pigmentation and abnormal tyrosinase trafficking is a feature of some melanomas (Halaban et al. 2002). Because it acts on L-3,4-dihydroxyphenylalanine (L-DOPA), tyrosinase has been linked to Parkinson's disease and other neurodegenerative diseases. Thus tyrosinase has been the subject of investigation in a broad range of contexts (Matoba et al. 2006).
Biochemical pathways
Fig. 1 Simplified diagram of early steps in the biochemical pathway leading to melanin production in mammals (modified from Schallreuter et al. 2007 and Chang 2009).
The biochemical pathway that leads to the production of melanin in mammals is complex (Chang 2009) and tyrosinase plays a key role in its regulation in the pathway's early steps (Schallreuter et al. 2007). In particular, the conversion of tyrosine to dopaquinone catalyzed by tyrosinase is the rate-limiting reaction in melanin synthesis because later steps occur spontaneously at physiological pH (Halaban et al. 2002). In today's lab we will investigate the kinetics of tyrosinase under varying conditions.
Because the substrate (tyrosine) is colorless and the product (dopachrome) is a colored substance, the progress of the reaction can be observed spectrophotometrically. The absorbance due to the presence of the dopachrome is directly proportional to the molar concentration of dopachrome (see the Introduction to Experiment 3) and since the cuvette contains a fixed volume, the absorbance is also directly proportional to the moles of dopachrome produced. So although the absorbance does not provide a direct value for moles of product it is a proxy for that quantity.
References
Chang, T-S. 2009. An updated review of tyrosinase inhibitors. International Journal of Molecular Sciences 10:2440-2475. http://dx.doi.org/10.3390/ijms10062440
Halaban, R., R.S. Patton, E. Cheng, S. Svedine, E.S. Trombetta, M.L. Wahl, S. Ariyan, and D.N. Herbert. 2002. Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway. Journal of Biological Chemistry 277:14821-14828. http://dx.doi.org/10.1074/jbc.M111497200
Matoba, Y., T. Kumagai, A. Yamamoto, H. Yoshitsu, and M. Sugiyama. 2006. Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. Journal of Biological Chemistry 281:8981-8990. http://dx.doi.org/10.1074/jbc.M509785200
Schallreuter, K.U., S. Kothari, B. Chavan, and J.D. Spencer. 2008. Regulation of melanogenesis - controversies and new concepts. Experimental Dermatology 17:395-4004. http://dx.doi.org/10.1111/j.1600-0625.2007.00675.x
