Pre-lab assignment 8
Name: Score: /5
1. CBL will recognize cystine and djenkolic acid as well as the native cystathionine as substrates in the beta-elimination reaction. What are the structures of these three substrates and what are the products of the enzyme catalyzed reaction?
2. Read through the background sections covering enzyme kinetics from the lab manual. What is the definition of Km? What are the Km values for the substrates; cystathionine, cystine and djenkolic acid?
3. What is the meaning of kcat? How is it calculated?
Notebook assignment. /5. We will begin working with CBL in a kinetic-type assay. In your notebook, create a specific procedure (solution concentrations etc.) or table to begin gathering experimental data.
Kinetic assay requirements:
1 mL buffer
50 mcL of 10 mM DTNB (indicator)
substrate concentrations: 10mM to 10 mM
Stock solution of cystine provided at 250 mM
CBL (remember [E] << [S], we will work next week to determine the "right" amount needed)
Remember in we need to measure the initial velocity (Vo) at a varitey of substrate concentrations. I would recommend a minimum of six reactions at substrate concentrations that vary from well below the Km to well above the Km.