Enzyme kinetics Flashcards
What is chymotrypsin?
Serine protease found in digestive system of mammals → contains a serine residue
arranged in three peptide chains linked by disulphide bridges
What organ secretes chymotrypsin and what is it secreted as?
Pancreas
Pro-eznyme chymotrypsinogen
What are proteases key for regulating?
protein maturation - removal of signal peptides
Degradation of ECM by migrating cells
General protein turnover
What are the requirements for recognition by chymotrypsin (give 3 examples)?
An aromatic side chain
e.g.’s phenylalanine, tyrosine or tryptophan
What are the requirements for recognition by chymotrypsin (give 3 examples)?
An aromatic side chain
e.g.’s phenylalanine, tyrosine or tryptophan
What is Km?
Michaelis constant - defined as the concentration of substrate at which a particular enzyme works at half of its maximal velocity
Low Km = weak binding
High Km = tight binding
What reaction does chymotrypsin catalyse?
Hydrolysis of GPNA generating N-glutaryl-L-phenyl alanine and ρ-nitroaniline
How is chymotrypsin activity detected?
Spectrometry -
ρ-nitroaniline obeys beer lambert law
follow cleavage of GPNA by detection of ρ-nitroaniline as it has significant absorbance at 410nm
What are the two protocols to measure production of ρ-nitroaniline ?
1: measure the production of p-nitroaniline as a function of time using several different concentrations of GPNA
2: GPNA solution and reaction buffer, pH 7.8 into cuvette
The cuvette contents were mixed and placed into a spectrophotometer set to measure absorbance at 410 nm.
The CAL button set the absorbance to zero.
chymotrypsin added, the contents mixed and the absorbance of the cuvette contents was recorded at timed intervals
What does the parameter Vmax tell you about the activity of chymotrypsin and how does this relate to the turnover number of an enzyme?
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).
What are the x and y axis of the Lineweaver-Burk plot showing respectively?
x axis - 1/[S] (Substrate concentration)
y axis - 1/V (velocity)
How do you find the Km from the Lineweaver-Burk plot?
-
Extrapolating the straight line graph until it crosses the x-axis gives a 1/[S] value of –1/KM
How do you find the Vmax from the Lineweaver-Burk plot?
the intercept on the y-axis
as [S] increases, 1/[S] tends towards zero, so that 1/Vmax is determined as the value of 1/V0 when 1/[S] = 0
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?
Indole → competitive inhibitor
Indole therefore increases Km
In contrast, the maximal velocity (Vmax) will be unaffected
How does the lineweaver burk plot change for competitive inhibition?
Higher Km and the same Vmax
steeper graph, same y intercept
