2.14 Enzyme Kinetics

Question 1

Which enzyme is used in the enzyme kinetics practical?

Answer 1

Chymotrypsin

Question 2

Why is chymotrypsin considered a serine protease?

Answer 2

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

Question 3

What does chymotrypsin do?

Answer 3

Cleaves a peptide bond with a preference for bulky side chains

Question 4

What is meant by Vmax

Answer 4

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

Question 5

What is Km?

Answer 5

The michaelis constant

Question 6

What is Km =?

Answer 6

1/2 V max

Question 7

What is meant by 1/2 V max?

Answer 7

Half the maximal velocity of the reaction

Question 8

Is the Km of hexokinase in muscle lower or higher?

Answer 8

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

Question 9

What is the Km of hexokinase in the liver?

Answer 9

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 10

what does the slope of the lineweaver burk plot show?

Answer 10

Km / Vmax

Question 11

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

Answer 11

-1/Km

Question 12

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

Answer 12

1/Vmax

Question 13

What are we measuring the absorbance of in the practical?

Answer 13

p-nitroaniline which is a bright yellow product

Question 14

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

Answer 14

410nm - this is because at this wavelength, the absorbance of GPNA is basically nothing and the absorbance of p-nitroaniline is much higher

Question 15

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

Answer 15

Adding a competitive inhibitor causes the Slope to become steeper - goes thorugh the same Y AXIS however the X axis intercept is changed and lies closer to 0

Question 16

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

Answer 16

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

Question 17

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

Answer 17

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

Question 18

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

Answer 18

Causes Vmax to decrease and Km to remain unchanged

Question 19

What is a competitive inhibitor?

Answer 19

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 20

What is a non-competitive inhibitor?

Answer 20

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

Question 21

What is the turnover number

Answer 21

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 22

What is the Kcat?

Answer 22

The turnover number

Question 23

What is the Kcat of chymotrypsin?

Answer 23

100

Question 24

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 24

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 25

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

Answer 25

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 26

What does a high Km value indicate

Answer 26

Weaker bonding as the substrate is only saturing at higher concentrations

Question 27

What does a low Km value indicate?

Answer 27

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

Question 28

What does a low Km value indicate?

Answer 28

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