enzyme kinetics

Question 1

• What is chymotrypsin?

- What organ secretes chymotrypsin and what is it secreted as?

Answer 1

Serine protease → contains a serine residue

Pancreas
Pro-eznyme chymotrypsinogen

Question 2

• What are the requirements for recognition by chymotrypsin (give 3 examples)?

Answer 2

An aromatic side chain (or bulky hydrophobic)

e.g.'s phenylalanine, tyrosine or tryptophan

Question 3

• Where does cleavage, done by the chymotrypsin, happen?

- What is GPNA?

Answer 3

Carboxyl side of the peptide bond (between CO and NH)

Artificial enzyme substrate known as N-Glutaryl-L-phenylalanine p-nitroanilide

Question 4

• What does the hydrolysis of GPNA catalysed by chymotrypsin generate?
• At what wavelength does p-nitroaniline have a significant absorbance in spectrophotometry?

Answer 4

N-glutaryl-L-phenyl alanine and the bright yellow product, p-nitroaniline

410

Question 5

• Define the Michaelis Constant (Km)

- How is the Km biochemically useful?

Answer 5

The concentration of substrate at which a particular enzyme works at half its maximal velocity

Allows us to compare the strength of Enzyme-substrate complexes

Question 6

• What does a low Km indicate?

- What does a high Km indicate?

Answer 6

Tight binding of a substrate to an enzyme

Indicative of weak binding

Question 7

• What does the parameter Vmax tell you about the activity of chymotrypsin
• How does this relate to the turnover number of an enzyme?

Answer 7

Vmax tells us the maximum velocity at which chymotrypsin can cleave peptide bonds

If we know the enzyme concentration used to derive Vmax, then by dividing Vmax by the enzyme concentration we can obtain the number of peptide bonds that chymotrypsin can cleave in a second
This is the turnover number (also known as Kcat).

Question 8

• What does the phrase ‘steady state’ mean in the initial phase of a reaction?

Answer 8

E-S complexes being formed and consumed at the same rate

Reaction velocity remains constant

Question 9

• What are the x and y axis of the Lineweaver-Burk plot showing respectively?

Answer 9

x axis - 1/[S] (Substrate concentration)

y axis - 1/V (velocity)

Question 10

• How do you find the Km from the Lineweaver-Burk plot?

- How do you find the Vmax from the Lineweaver-Burk plot?

Answer 10

Extrapolating the straight line graph until it crosses the x-axis gives a 1/[S] value of –1/KM

As [S] increases, 1/[S] tends towards zero, so that 1/Vmax is determined as the value of 1/V0 when 1/[S] = 0 
i.e. the intercept on the y-axis

Question 11

• Chymotrypsin activity is inhibited by the small molecule indole which binds within the active site of chymotrypsin. What do you think would be the effects of indole upon the parameters Km and Vmax for chymotrypsin?

Answer 11

Indole → competitive inhibitor as it binds within the active site of chymotrypsin

Indole therefore increases Km

In contrast, the maximal velocity (Vmax) will be unaffected

Question 12

• Why is the Vmax unaffected in competitive inhibition?

- Why is the Km increased during competitive inhibition?

Answer 12

Because increasing amounts of substrate can swamp the inhibitor (present in fixed concentration)
Allowing the enzyme to effectively not see the inhibitor at high substrate concentrations

It takes more substrate to get the competitively inhibited reaction to Vmax/2

Question 13

• In non-competitive inhibition, why does the Km not change?

Answer 13

Km is a measure of the affinity of the enzyme for its substrate and this can only be measured by active enzyme

The fixed amount of inactive enzyme in non-competitive inhibition does not affect the Km and the Km, therefore is unchanged