Enzyme Kinetics Flashcards
Which enzyme is used in the enzyme kinetics practical?
Chymotrypsin
What is chymotrypsin?
A serine protease found in the digestive system of mammals
Arranged in 3 peptide chains (A,B and C) linked by disulphide bridges
Why is chymotrypsin considered a serine protease?
Serine is essential for the functioning of the protease, if it is not present the enzyme will not function
What does chymotrypsin do?
Cleaves a peptide bond with a preference for bulky side chains
Proteases are also key for regulation of other processes, what are these other processes?
Protein maturation (e.g. removal of signal peptides)
Degradation of ECM by migrating cells
General protein turnover
What is chymotrypsin’s specificity?
bulky hydrophobic side chains
- the bond cyan is cleaved, between the phenylalanine (Phe) and asparagine (Asn)
What is meant by Vmax
Vmax is the maximum rate of reaction - the highest speed the reaction can proceed at based on how much enzyme you have
What is Km?
The Michaelis constant
= defined as the concentration of substrate at which a particular enzyme works at half its maximum velocity
Why is Km useful?
useful as a means of comparing the strength of enzyme-substrate complexes
What is Km =?
1/2 V max
What is meant by 1/2 V max?
Half the maximal velocity of the reaction
Is the Km of hexokinase in muscle lower or higher?
Hexokinase is working flat out all the time, and therefore will have a LOWER Km - this indicates tight bonding
What is the Km of hexokinase in the liver?
4 - much higher than that in the muscle - this is because in the liver, there are variable concentrations of glucose at different points in time and the higher Km allows it to respond to different concentrations of glucose
what does the slope of the lineweaver burk plot show?
Km / Vmax
what does the x intercept of the lineweaver burk plot show?
-1/Km
what does the y intercept of the lineweaver burk plot show?
1/Vmax
What are we measuring the absorbance of in the practical?
p-nitroaniline which is a bright yellow product
What wavelength do we measure the absorbance of p-nitroaniline at?
410nm - this is because at this wavelength, the absorbance of GPNA is basically nothing and the absorbance of p-nitroaniline is much higher
it allows us to monitor production formation
Draw a curve which shows the effects of adding a competitive inhibitor to a substrate and enzyme solution
Adding a competitive inhibitor causes the Slope to become steeper - goes through the same Y AXIS however the X axis intercept is changed and lies closer to 0
Draw a curve which shows the effects of adding a non-competitive inhibitor to a substrate and enzyme solution
Adding a non-competitive inhibitor affects the Y INTERCEPT - INCREASES, however the X INTERCEPT remains constant
What affect does a competitive inhibitor have on V max and Km?
causes V max to be unchanged, and Km to increase with a competitive inhibitor
What affect does a non-competitive inhibitor have on Vmax and Km?
Causes Vmax to decrease and Km to remain unchanged
What is a competitive inhibitor?
A molecule which binds to the enzyme instead of the substrate therefore impairing its funcion - simply adding enough substrate should outcome the competitive inhibitor
What is a non-competitive inhibitor?
Molecule which binds the enzyme themselves, outside the active site and induces a conformational change such that enzyme function is inhibited
What is the turnover number
the number of molecules an enzyme can turnover in a given period of time (usually 1 second) - essentially equivalent to the number of bonds an enzyme can cleave in a second
What is the Kcat?
The turnover number
What is the Kcat of chymotrypsin?
100
What is steady state?
During the initial phase of the reaction, as long as the reaction velocity remains constant, the reaction is in a steady state, with ES being formed and consumed at the same rate.
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?
Recall that molecules binding within the active site of an enzyme compete with the substrate for binding to the enzyme. It could therefore be reasonably assumed that indole competes with substrate for binding to chymotrypsin and therefore higher substrate concentrations are needed to obtain a half-maximal velocity. Indole therefore increases KM. In contrast, the maximal velocity (Vmax) will be unaffected.
What does the parameter KM tell you about the interaction of chymotrypsin with GPNA?
A KM of around 1-2 mM suggests that chymotrypsin binds GPNA with high affinity. Recall that the lower the KM value, the higher the affinity of the enzyme for its substrate.
What does a high Km value indicate?
Weaker bonding as the substrate is only saturing at higher concentrations
What does a low Km value indicate?
Tighter bonding between the enzyme and substrate - at lower concentrations the enzyme is still working efficiently
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).
With a turnover number of 100 bonds cleaved per second, chymotrypsin is not particularly slow!
Given that the turnover number of chymotrypsin is approximately 100/s, how does it compare to those of the enzymes lysozyme and carbonic anhydrase?
Lysozyme is relatively slow, cleaving a single bond only every 2 seconds. In contrast, carbonic anhydrase is one of the fastest enzymes known, catalysing upwards of a million reactions per second
What does the parameter KM tell you about the interaction of chymotrypsin with GPNA?
A KM of around 1-2 mM suggests that chymotrypsin binds GPNA with high affinity. Recall that the lower the KM value, the higher the affinity of the enzyme for its substrate.
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?
Recall that molecules binding within the active site of an enzyme compete with the substrate for binding to the enzyme. It could therefore be reasonably assumed that indole competes with substrate for binding to chymotrypsin and therefore higher substrate concentrations are needed to obtain a half-maximal velocity. Indole therefore increases KM. In contrast, the maximal velocity (Vmax) will be unaffected.
What would a lineweaver-burk plot look like for non-competitive inhibitors?
What would a lineweaver-burk plot look like for competitive inhibitors?
