Rapid Reactions

Question 1

What is Vmax?

Answer 1

Initial rate of a reaction will increase as the substrate concentration increases up to a maximum value

This is when all the enzyme active sites are occupied by the substrate

Question 2

What does alcohol dehydrogenase catalyse (ADH)?

Answer 2

The reversible interconversion of ethanol and acetldehyde (ethanal)

Ethanol + NAD+ <—> Acetaldehyde + NADH + H+

Question 3

What is the function of YADH?

Answer 3

An enzyme in yeast that normally functions in the direction of ethanol formation during fermentation

Question 4

Function of ADH

Answer 4

In mammals, ethanol is metabolised in the liver where cytosolic ADH converts ethanol into acetaldehyde which in turn is oxidised by the enzyme aldehyde dehydrogenase (ALDH) to acetate

Acetate the enters the TCA cycle as acetyl-CoA for further breakdown

Question 5

What is the importance of the different ALDH genes?

Answer 5

There are 5 different genes

A deficiency in the low Km ALDH (ALDH2) is common in the Asian population and individuals deficient in this enzyme cannot tolerate alcohol since they cant rapidly convert acetaldehyde to acetate

Question 6

Summary of the experiment

Answer 6

• Set up a series of tubes containing graded concentrations of the substrate = [S]
• Add, at time zero, a fixed amount of the enzyme preparation
• Measure the initial reaction velocity (V0) which is the concentration of the product (NADH) formed over a short initial time of the reaction
• The product absorbs light so this can easily be measured using a spectrophotometer
• Early in the run, when the amount of substrate is in excess to the amount of enzyme, the observed rate is the initial velocity of V0
• Determine Vmax and Km by measuring the initial rate of reaction, V0, with increasing substrate concentration

Question 7

At what wavelength does NADH absorb light?

Answer 7

340 nm

The absorbance at 340 nm will increase with time

Question 8

How is the initial rate of reaction measured using absorbance?

Answer 8

V0 = ∆c / ∆t

Units are M min-1

Question 9

How is the beer-lambert law used to convert ∆A per min to the ∆C?

Answer 9

∆A / ∆t = e x ∆c x L /∆t

Question 10

How is the dilution factor calculated?

Answer 10

Total volume divided by volume of X

Question 11

How is the concentration of ethanol calculated?

Answer 11

Dividing the stock volume by the dilution factor then x1000