Enzyme Activity

Question 1

Define transition state

Answer 1

High energy intermediate that lies between substrate and product

Question 2

Define activation energy

Answer 2

Minimum energy substrate must have to allow reaction

Question 3

Name 2 ways to increase the rate of reaction

Answer 3

Temperature - increase number o molecules with activation energy
Concentration - increase chance of molecular collisions

Question 4

Define enzymes

Answer 4

Biological catalysts that increase the rate of reaction by lowering the activation energy - facilitate formation of the transition state

Question 5

List some important features of enzymes

Answer 5

Highly specific
Unchanged after reaction
Do not affect reaction equilibrium 
Increase rate of reaction
Proteins
May require associated cofactors

Question 6

What is the clinical relevance of enzymes?

Answer 6

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

Question 7

Define active site

Answer 7

The active site of an enzyme is the place where substrates bind and where the chemical reaction occurs

Question 8

How much of the enzyme does that active site occupy?

Answer 8

1. The active site occupies a small part of the enzyme
   Most enzymes are >100 aa but active site is only a few aa
   Most of enzyme acts as a scaffold to create the active site

Question 9

What is the active site formed from?

Answer 9

The active site is formed by amino acids from different parts of the primary sequence

Question 10

Describe the shape of the active site and describe the 2 models of substrate fit

Answer 10

Active sites are clefts or crevices
Substrate molecules are bound in a cleft or crevice that usually excludes water

Active sites have a complementary shape to the substrate
“Lock and Key” hypothesis

Binding of substrates can induce changes in the conformation “Induced fit” hypothesis - The active site only forms a complementary shape after binding of the substrate

Question 11

How are substrates bound to enzymes?

Answer 11

Substrates are bound to enzymes by multiple weak bonds
All types of non-covalent bonds
Binding must not be too tight!

Question 12

What is V0?

Answer 12

Initial rate of reaction

Question 13

What happened to enzyme activity as temperature increases?

Answer 13

Increases up to optimum
Then enzyme denatured
Activity decreases past optimum

Question 14

Describe the shape of a graph of reaction velocity against substrate concentration

Answer 14

Rectangular hyperbola - it reaches a maximal velocity

Question 15

What is the Michaelis-Menten model for enzyme catalysis?

Answer 15

The model proposes that a specific complex between the enzyme and the substrate is a necessary intermediate in catalysis.

V0 = Vmax[S]/Km+[S]

Predicts that a plot of V0 against [S] will be a rectangular hyperbola

NOT ALL ENZYMES OBEY THIS MODEL

Question 16

Define Vmax

Answer 16

Maximal rate when all enzyme active sites are saturated with substrate

Question 17

Define Km

Answer 17

Substrate concentration that gives half maximal velocity
Km = Michaelis constant (M)
M = moles/litre

Question 18

What is the significance of Km values?

Answer 18

Km values give a measure of the affinity of an enzyme for it’s substrate

Low Km = high affinity for substrate
High Km = low affinity for substrate

Question 19

What is the significance of Vmax/V0 values?

Answer 19

Vmax/V0 values are rates
Measured in amounts per unit time

Often expressed as a standardised rate: per litre (L) of serum or per gram (g) of tissue

The rate of an enzyme catalysed reaction is proportional to the concentration of enzyme

Double the amount of enzyme – double the rate BUT – not the standardised rate

Question 20

What is 1 unit?

Answer 20

1 unit = the amount of enzyme that converts 1mmol of product per min under standard conditions

Question 21

What is the Lineweaver-Burk plot

Answer 21

Michaelis-Menten equation can be rearranged to give a linear plot

V0 = Vmax[S]/Km+[S]

Y axis = 1/V
X axis = 1/[S]
Gradient = Km/Vmax
Y intercept = 1/Vmax
X intercept = -1/Km

Allows for easy estimation of Km and Vmax

Question 22

What are enzyme inhibitors?

Answer 22

Molecules that slow down or prevent an enzyme reaction

- including many drugs

Question 23

What are the types of enzyme inhibitors?

Answer 23

1. Irreversible: Bind very tightly, generally form
   covalent bond(s)
   Example: nerve gases, such as sarin
2. Reversible: Non-covalent; can freely dissociate.
   i) Competitive – binds at active site.
   ii) Non-competitive – binds at another site on the enzyme - the allosteric site

Question 24

Describe competitive inhibition

Answer 24

The competitive inhibitor resembles the substrate and binds to the active site, reducing the proportion of enzyme molecules bound to the substrate.

Question 25

How does competitive inhibition affect Km and Vmax?

Answer 25

Adding enough substrate will always overcome the effect of the inhibitor, so no effect on Vmax

Because the inhibitor competes with the substrate for the active site Km increases

Slower speed to reach same Vmax

Question 26

Describe non-competitive inhibition

Answer 26

Non-competitive inhibitor binds at an alternative site (allosteric site) and decreases the turnover number of the enzyme

Question 27

How does non-competitive inhibition affect Km and Vmax?

Answer 27

Lowers Vmax but Km unaffected

Same speed reaction but Vmax is lower