Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course

Abstract

A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.