Introduction to Enzymes

Question 1

What are enzymes?

Answer 1

• proteins

- biological catalysts

Question 2

How do enzymes increase rate of reaction?

Answer 2

• provide a pathway of lower activation energy to get reactants to products by stabilising the transition state

Question 3

How do enzymes work?

Answer 3

• form complexes with their substrates (binding), providing a unique microenvironment for reaction to proceed, the active site
• not chemically altered in the reaction
• very high specificity

Question 4

Explain how activity of enzymes can be regulated

Answer 4

enzymes can be modified to either be activated or inhibit the enzymes activity

Question 5

Why are enzymes important in medicine?

Answer 5

almost every chemical reaction in a cell is catalysed by a specific enzyme

• disease - enzyme deficiencies
• diagnosis - measure enzyme levels
• drug therapy - many drugs are enzyme inhibitors
• basic research - there remains unclassified enzymes

Question 6

What is the function of lysozyme?

Answer 6

catalyses the cutting of polysaccharide chains

Question 7

What happens as the substrate concentration increases?

Answer 7

reaction velocity increases

Question 8

Why is product accumulation not linear? ( why is V0 initial reaction velocity the only valid parameter)

Answer 8

• substrate concentration falls as it turns to product
• products may inhibit enzyme
• products may inhibit enzyme
• enzyme may denature
• reverse reaction becomes more favourable

Question 9

How is enzyme activity measured?

Answer 9

increasing the substrate concentration and measuring the accumulation of products over time
the reaction reaches a point where it can no longer increase - the maximum Vmax is reached

Question 10

What is Vmax?

Answer 10

when the maximum rate of reaction is reached - this increases as substrate concentration increases

Question 11

Since Vmax is hard to achieve how is it calculated?

Answer 11

• plotting a double reciprocal of the date - Lineweaver-burke plot
• 1/V against 1/[S]
• y intercept = 1/Vmax

Question 12

What is Km?

Answer 12

the substrate concentration required for half of max velocity (Vmax)

Question 13

Why is Km useful?

Answer 13

it describes the ease at which the substrate binds to enzyme “affinity”

• Km is inversely proportional to the affinity (low Km = high affinity)
• only a low concentration of substrate is requires to saturate the enzyme at low Km

Question 14

What are enzyme inhibitors?

Answer 14

chemicals that interfere with enzyme reactions - many drugs are inhibitors

Question 15

What are the classification of enzyme inhibitors?

Answer 15

• irreversible (inactivators)

- Reversible (competitive or allosteric)

Question 16

What is irreversible inhibition?

Answer 16

inhibitor react with enzyme. and forms a covalent adduct with the protein - inactivation of the enzyme is irreversible

Question 17

What are two examples of irreversible inhibition?

Answer 17

• organophosphates (found in pesticides) - damages the enzyme acetylcholinesterase (AChE) which is responsible for degrading the neurotransmitter acetylcholine(ACh.) After inhibition ACh accumulates throughout the nervous system, resulting in overstimulating of receptors
• Aspirin (acetylsalicylic acid) - antipyretic, anti-inflammatory, analgesic that inhibits cyclooxyrgenase (COX-1) which is the enzyme that catalyses an inflammatory reaction - aspirin reacts with a serine residue close to the active site inhibiting the enzyme

Question 18

What is competitive inhibition?

Answer 18

inhibitor competes with the substrate for the active site of the enzyme - the inhibitor has a similar structure to that of the normal substrate - the drug occupies the active site but leaves it unchanged - this slows down the action of the enzyme

Question 19

What us an example of competitive inhibition?

Answer 19

Sulphonamides inhibit the enzyme involved in folic acid synthesis in bacteria - similar structure to the substrate 4-aminobenzoic acid- meaning they work as good antibiotics as they starve the bacteria of essential folic acid

Question 20

How do competitive inhibitors alter the kinetics of an enzyme?

Answer 20

• inhibitor binds reversibly to the active site
• inhibitor and substrate compete for the active site
• therefore a higher concentration of substrate is needed to reach Vmax resulting in an increase in Km (Vmax unchanged)

Question 21

What is allosteric inhibition?

Answer 21

alleosteric inhibitors bind to the enzyme at the same time as the substrate (never to active site) - making the enzyme and enzyme-substrate complex inactive because the active site changes conformation

• the inhibitor cannot be driven from the enzyme by higher substrate concentration
• Vmax decreases - Km often (not always) increases

Question 22

What are the two types of allosteric inhibition?

Answer 22

• mixed inhibition - affects substrate binding and activity - inhibition can be reduced by increasing substrate concentration - Vmax decreases due to inhibition - Km increases as substrate affinity is reduced
• non-competitive inhibition- affects activity but not substrate binding - Vmax decreases due to reduced efficiency of the reaction - Km is unchanged as substrate affinity is unchanged

Question 23

What is an example of allosteric inhibition?

Answer 23

phosphofructokinase (PFK) catalyses the transfer of phosphate from ATP to fructose 6-phosphate which is an important step in glycolysis - PFK binds to ATP at both the active site and inhibitory site - in the presence of high levels of ATP, the inhibitory site is occupied and fructose 6-phosphate binding is affected - so glycolysis does not proceed when ATP is not needed