Enzyme kinetics

Question 1

What is Vmax?

Answer 1

Maximum enzyme velocity - approached asymptotically as [S] becomes large

Question 2

What is Km?

Answer 2

The substrate concentration at which the rate of reaction is exactly half that of the Vmax.

Question 3

How is the rate of enzyme catalysis calculated?

Answer 3

V0 = Vmax * ([s]/([s] +Km)

Question 4

Why can’t we measure Vmax directly and how is it done then?

Answer 4

• Vmax can be difficult to determine with accuracy directly
• Lineweaver and Burk devised a method of plotting the data as reciprocals.
• The initial velocity (V0) of a series of reactions are
  calculated for a range of substrate concentrations [S].
• As [S] increases 1/[S] tends towards zero, so that 1/Vmax is determined as the value of 1/V0 when 1/[S] = 0
• Extrapolating the straight line graph until it crosses the other axis
  gives a 1/[S] value of –1/KM.

Question 5

What is chymotrypsin?

Answer 5

• Secreted by the pancreas as an inactive form
• Undergoes proteolysis in the duodenum, to form active chymotrypsin which can hydrolyse peptide bonds and aid protein digestion.

Question 6

What is the overview of the experiment?

Answer 6

Adding an artificial substrate to this enzyme that when cleaved forms a bright yellow product - p nitrolaniline. Reaction can be followed with spectrophotometry as it has a significance absorbance at 410 nm unlike the substrate
Reaction will be carried out at different concentrations of GPNA (substrate)

Question 7

When can we use the Michealis -Menten kinetics and Lineweaver-Burk plot?

Answer 7

When GPNA is in excess, the steady-state reaction phase catalysed by chymotrypsin conforms to Michaelis-Menten kinetics and can be analyzed with a Lineweaver-Burk plot.

Question 8

How can Vmax and Km be determined by plotting graphs and using calculations?

Answer 8

1. Make a graph plotting time vs absorbance for all the GPNA concentrations
2. To find concentrations at particular points use the Beer Lambert law (exitinction coefficient is 8.8 and length is 1cm)
3. For each GPNA solution work out V0
4. make a graph of 1/V0 versus 1/[S] (Lineweaver Burk plot)
5. 1/Vmax = y intercept
   - 1/Km = x intercept