Enzyme Kinetics

Question 1

Which enzyme is used in the enzyme kinetics practical?

Answer 1

Chymotrypsin

Question 2

What is chymotrypsin?

Answer 2

A serine protease found in the digestive system of mammals

Arranged in 3 peptide chains (A,B and C) linked by disulphide bridges

Question 3

Why is chymotrypsin considered a serine protease?

Answer 3

Serine is essential for the functioning of the protease, if it is not present the enzyme will not function

Question 4

What does chymotrypsin do?

Answer 4

Cleaves a peptide bond with a preference for bulky side chains

Question 5

Proteases are also key for regulation of other processes, what are these other processes?

Answer 5

Protein maturation (e.g. removal of signal peptides)

Degradation of ECM by migrating cells

General protein turnover

Question 6

What is chymotrypsin’s specificity?

Answer 6

bulky hydrophobic side chains
- the bond cyan is cleaved, between the phenylalanine (Phe) and asparagine (Asn)

Question 7

What is meant by Vmax

Answer 7

Vmax is the maximum rate of reaction - the highest speed the reaction can proceed at based on how much enzyme you have

Question 8

What is Km?

Answer 8

The Michaelis constant
= defined as the concentration of substrate at which a particular enzyme works at half its maximum velocity

Question 9

Why is Km useful?

Answer 9

useful as a means of comparing the strength of enzyme-substrate complexes

Question 10

What is Km =?

Answer 10

1/2 V max

Question 11

What is meant by 1/2 V max?

Answer 11

Half the maximal velocity of the reaction

Question 12

Is the Km of hexokinase in muscle lower or higher?

Answer 12

Hexokinase is working flat out all the time, and therefore will have a LOWER Km - this indicates tight bonding

Question 13

What is the Km of hexokinase in the liver?

Answer 13

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

Question 14

what does the slope of the lineweaver burk plot show?

Answer 14

Km / Vmax

Question 15

what does the x intercept of the lineweaver burk plot show?

Answer 15

-1/Km

Question 16

what does the y intercept of the lineweaver burk plot show?

Answer 16

1/Vmax

Question 17

What are we measuring the absorbance of in the practical?

Answer 17

p-nitroaniline which is a bright yellow product

Question 18

What wavelength do we measure the absorbance of p-nitroaniline at?

Answer 18

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

Question 19

Draw a curve which shows the effects of adding a competitive inhibitor to a substrate and enzyme solution

Answer 19

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

Question 20

Draw a curve which shows the effects of adding a non-competitive inhibitor to a substrate and enzyme solution

Answer 20

Adding a non-competitive inhibitor affects the Y INTERCEPT - INCREASES, however the X INTERCEPT remains constant

Question 21

What affect does a competitive inhibitor have on V max and Km?

Answer 21

causes V max to be unchanged, and Km to increase with a competitive inhibitor

Question 22

What affect does a non-competitive inhibitor have on Vmax and Km?

Answer 22

Causes Vmax to decrease and Km to remain unchanged

Question 23

What is a competitive inhibitor?

Answer 23

A molecule which binds to the enzyme instead of the substrate therefore impairing its funcion - simply adding enough substrate should outcome the competitive inhibitor

Question 24

What is a non-competitive inhibitor?

Answer 24

Molecule which binds the enzyme themselves, outside the active site and induces a conformational change such that enzyme function is inhibited

Question 25

What is the turnover number

Answer 25

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

Question 26

What is the Kcat?

Answer 26

The turnover number

Question 27

What is the Kcat of chymotrypsin?

Answer 27

100

Question 28

What is steady state?

Answer 28

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.

Question 29

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 29

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.

Question 30

What does the parameter KM tell you about the interaction of chymotrypsin with GPNA?

Answer 30

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.

Question 31

What does a high Km value indicate?

Answer 31

Weaker bonding as the substrate is only saturing at higher concentrations

Question 32

What does a low Km value indicate?

Answer 32

Tighter bonding between the enzyme and substrate - at lower concentrations the enzyme is still working efficiently

Question 33

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

Answer 33

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!

Question 34

Given that the turnover number of chymotrypsin is approximately 100/s, how does it compare to those of the enzymes lysozyme and carbonic anhydrase?

Answer 34

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

Question 35

What does the parameter KM tell you about the interaction of chymotrypsin with GPNA?

Answer 35

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.

Question 36

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 36

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.

Question 37

What would a lineweaver-burk plot look like for non-competitive inhibitors?

Answer 37

Question 38

What would a lineweaver-burk plot look like for competitive inhibitors?

Answer 38