L4 - Enzyme Kinetics

Question 1

Define the transition state

Answer 1

The high energy intermediate that lies between the substrate and the product

Question 2

Define the activation energy

Answer 2

The minimum energy the substrate must have to allow the reaction

Question 3

What are enzymes?

Answer 3

Biological catalysts that increase the rate of reaction by lowering the activation energy.
They do this by facilitating the formation of the transition state

Question 4

What are important features of enzymes?

Answer 4

Specific
Unchanged after the reaction
DO NOT AFFECT THE REACTION EQUILIBRIUM
Increase the rate of reaction
Protein (with one or two exceptions)
May require associated cofactors

Question 5

Why are we interested in enzymes?

Answer 5

Inheritable genetic disorders
Overactive enzymes can cause disease
Measurement of enzyme activity for diagnosis
Inhibition of enzymes by drugs

Question 6

Define an active site

Answer 6

A place in the enzyme where substrates bind and where chemical reactions occur

Question 7

What are the features of an active site?

Answer 7

1. A small part of the enzyme - few aa
2. Formed from aa from different parts of the sequence
3. Are clefts or crevices - usually exludes water
4. Complimentary shape to substrate - lock and key hypothesis

Question 8

What is the lock and key hypothesis?

Answer 8

Active sites have a complimentary shape to the substrate

Question 9

What is the induced fit hypothesis?

Answer 9

Binding of the substrate can induce changes in conformation. Therefore the active site only forms a complimentary shape after binding of the substrate

Question 10

How does an enzyme bind its substrate?

Answer 10

Not too tightly with multiple non-covalent bonds (any type)

Question 11

Explain the process through which enzymes work

Answer 11

1. S alone
2. E binds the substrate to form ES complex (lower E)
3. ES transition state occurs (high energy state)
4. P formed, alone

Question 12

What is Vo?

Answer 12

The initial rate of reaction. This is the only time during the reaction where the concentration of substrate is known

Question 13

What is the shape of a graph that plots reaction velocity (y axis) against substrate concentration (x axis)?

Answer 13

Rectangular hyperbola - as the enzyme-catalysed reaction reaches a maximum velocity and plateaus

Question 14

What does the Michaelis-Menten model for enzyme catalysis propose?

Answer 14

That a specific complex between the enzyme and the substrate is a necessary intermediate in catalysis.
It predicts that a plot of Vo versus [S] will be a rectangular hyperbola.
NOT ALL ENZYMES OBEY THIS MODEL e.g. those that follow cooperativity like haemoglobin

Question 15

What does Km and Vmax mean?

Answer 15

Km = Substrate concentration that gives half maximal velocity
Vmax = Maximum rate when all enzymes in active sites are saturated with substrate (this will vary depending on the concentration of the enzyme)

Question 16

What does a high Km or low Km tell you about the enzyme and its substrate?

Answer 16

High Km - enzyme has a low affinity for its substrate

Low Km - enzyme has a high affinity for its substrate

Question 17

Hexokinase and glucokinase are enzyme which catalyse the same reaction (glucose -> glucose-6-phosphate). What is the difference between them?

Answer 17

Glucokinase (present in liver) has a higher Km than hexokinase (in all tissues), therefore glucokinase is only active at high concentrations of glucose - switching on glycolysis in the liver after feeding

Question 18

The Michaelis-Menten equation can be rearranged to give a linear plot. What is its name?

Answer 18

The Lineweaver-Burk plot

Question 19

In the Lineweaver Burk plot. What is the the y axis and x axis intercepts give you?

Answer 19

Y axis: 1/Vmax

X axis: -1/Km

Question 20

What are enzyme inhibitors?

Answer 20

Molecules that slow down or prevent an enzyme reaction

Question 21

What types of enzyme inhibitors are there?

Answer 21

1. Irreversible - generally form covalent bonds
2. Reversible - non-covalent, can freely dissociate
   (i) Competitive
   (ii) Non-competitive

Question 22

What are competitive inhibitors and how do they affect a reaction?

Answer 22

Resemble the substrate and bind to the active site of enzyme, reducing the proportion of enzyme bound to the substrate. Affect Km but not Vmax

Question 23

What are non-competitive inhibitors and how do they affect a reaction?

Answer 23

Bind at another site on the enzyme and decreases the turnover number of an enzyme. This does not effect the affinity that an enzyme has for its substrate (Km) but prevents it from entering the active site - effectively lowering the concentration of effective enzyme. Affect Vmax but not Km

Question 24

Why is Vmax not affected by competitive inhibition?

Answer 24

Adding enough substrate will always overcome the effects of the inhibitor and so have no effect on Vmax

Question 25

Why is the Km affected by competitive inhibition?

Answer 25

Because the competitive inhibitor binds to the active site, it competes for the active site with the substrate. Therefore a higher concentration of substrate is needed to achieve half the maximum velocity - the Km is increased