Purpose
What are the kinetic mechanisms of tyrosinase inhibitors? Over the next two weeks, we will characterize the mechanism of some inhibitors by discovering how these chemicals inhibit the enzyme kinetic parameters Km and Vmax. This week we will first perform the positive control measurement of Km and Vmax in the absence of inhibitors. Next week we will measure the effects of the inhibitors. In particular, Km is an intrinsic parameter that does not depend on the quantity of active enzyme and this thus comparable between groups and can be compared to literature values.
This lab will be divided into three sections; in Part A, you will determine the optimal enzyme amount for a fast, linear initial reaction rate. In Part B, you will determine what range of substrate concentration will allow you to calculate the Km of tyrosinase. Finally, in Part C, you will combine the results of A and B and construct an experiment that will monitor tyrosinase reaction rate as a function of substrate concentration.
Objectives
Upon successful completion of this lab, you will be able to:
- Design and execute protocols for determining enzyme kinetic parameters.
- Make accurate serial dilutions.
- Analyze the effect of changing either enzyme or substrate concentration on initial reaction rates.
- Construct Eadie-Hofstee plots and determine Km, Vmax and Vmax/Km parameters.
- Calculate standard error for trendline parameters using the LINEST arrayed equation in Excel.
- Locate Km values in the published literature.
- Compare Km to published literature values.
Pre-lab Preparation
Read the assigned reading (including this lesson) completely
Pass the prelab quiz with at least 70% by midnight before your lab
Outline the purpose and procedures sections in your notebook