RBM2560 - Enzyme Kinetics: Alcohol Dehydrogenase (ADH) - Medical Science Assignment Help

Assignment Task

Enzyme kinetics: alcohol dehydrogenase (ADH)

Read through this practical carefully. Then, fill out the tables of volumes of substrate (ethanol) (for substrate assay) to be added in the method. It would be good to prepare this prior to the lab and have it checked by your peers and lab demonstrator before setting up the assays.
Please ensure that you are prepared for the practical class as the reactions you prepare will be used by your classmates and precision and accuracy are important in these assays.

Introduction
Alcohol dehydrogenase is a zinc metalloenzyme of broad specificity. It can be obtained from a wide range of cells and tissues such as yeast and horse liver. The enzyme has Zn2+ ions in its catalytic site. The yeast enzyme is a tetramer of Mr 145,000. Each chain can bind one NAD+ and one Zn2+.
Binding of nucleotide cofactor (NAD+/NADH) causes conformational change in the enzyme that enables ethanol (or acetaldehyde in the reverse reaction) to bind the enzyme.
The liver enzyme is a dimer with various isozymes. The zinc ion is located at the bottom of a hydrophobic pocket. It is ligated to the main enzyme by thiol sulfurs of cysteine groups on the dehydrogenase and also by a nitrogen. The thiol groups are critical for the activity of the enzyme. The fourth ligand is a water molecule, hydrogen bonded to the hydroxyl group of a serine. The nicotinamide ring of NAD+ is bound close to the zinc ion.

Objectives:
The objectives of this practical are:
• To measure the activity of yeast alcohol dehydrogenase (ADH) at various pH and substrate concentrations.
• To estimate the optimum pH of yeast ADH.
• To determine the KM and Vmax of yeast ADH for ethanol.

Materials
Technical staff: Please note NAD+ and alcohol dehydrogenase should be freshly prepared just before the practical class and stored on ice.

1 mL NADH standard solution (0.5 mM)
500μL NAD+ (6 mM stock)
500μL 1 M C2H5OH = ethanol (1M in dH2O) (σ = 0.79 g/mL)
500μL of 0.1 M C2H5OH
2 mL buffer (0.1 M sodium phosphate, pH 8.0)
500μL buffer (0.1 M sodium phosphate, pH 4.5)
500μL buffer (0.1 M sodium phosphate, pH 5.0)
500μL buffer (0.1 M sodium phosphate, pH 6.0)
500μL buffer (0.1 M sodium phosphate, pH 10.0)
4 mL alcohol dehydrogenase (36.6μg /mL) (need to check with technical staff as to what concentration is available on the day)
5 mL ddH2O

Controls
You will have to include controls in this enzyme kinetics practical class and in most other experimental work you do in the future – so read this section carefully. Ask you demonstrator or lecturer for advice if you do not understand what a control is and why you use controls in experiments.
What are experimental controls and why have them? Perhaps the best way to answer this question is to look at examples of controls and how they are used. In today’s experiments you you will be determining enzyme activity by measuring the amount of product (NADH) made as a result of enzymic reduction of NAD+. However, to determine the amount of NADH (N) produced by the enzyme during a reaction it is no use simply measuring the mount of NADH present at the end of the reaction, as there will probably have been some NADH present at the start! Thus the NADH present at the end of the reaction is made up of what was there at the start and what has been produced by the action of the enzyme!

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