CBI 6: Enzymes and Kinetics of Biocatalysis

Question 1

Give an example of an enzyme that is not a protein

Answer 1

• ribozymes: they are enzymes comprised of RNA

Question 2

Describe this reaction profile

Answer 2

Question 3

Describe the enzyme mechanism

Answer 3

• the substrate (S) binds to a specific active site of the enzyme (E), forming an enzyme-substrate complex (ES)
• interactions between the substrate(s) and the functional groups in the active site of the enzyme, lower the activation energy by providing an alternative reaction pathway
• this accelerates the reaction process as less energy is required to reach the transition state
• following the formation of ES, the reaction mechanism will form the product (P) and free enzyme (E)
• the enzyme is released and ready to bind to more substrate

Question 4

How do you name enzymes?

Answer 4

• each enzyme is assigned four number that indicate their classification

Question 5

What database can you use to view a metabolic pathway and enzymes?

Answer 5

• KEGG PATHWAY

Question 6

Answer 6

Question 7

Describe this graph of an enzyme-catalysed reaction without an inhibitor present

Answer 7

• the rate of reaction at low substrate concentrations (so the initial velocity) is directly proportional to the concentration of the substrate
• so initially first order
• at high substrate concentration, the reaction kinetics resemble zero-order kinetics
• the reaction rate is unaffected by (any further increase in) substrate concentration

Question 8

What is V_max?

Answer 8

• this is the maximum velocity of an enzyme
• the enzyme is still converting substrate into products but the reaction mechanism has been reached
• in an enzyme-catalysed reaction without an inhibitor, the enzyme is still converting substrate into products, but the reaction equilibrium has been reached

Question 9

How do you mathematically describe an enzymatic reaction where a single substrate is converted to a single product?

Answer 9

• enzyme (E) combines with substrate (S) to form the enzyme-substrate (ES) complex, with a rate constant k₁
• the ES has then two possible fates:
  1. dissociate to E and S, with a rate constant k_-1
  2. proceed and form product (P) with rate constant k₂
• k₂ is commonly described as k_cat

Question 10

What is the Michaelis-Menten equation?

Answer 10

• When substrate concentration = K_M , reaction velocity is 1/2 V_max , meaning have the enzyme active sites are filled at that moment

Question 11

Answer 11

Question 12

Is V_max reached?

Answer 12

• V_max is approached but never reached

Question 13

What does the value of K_M tell us?

Answer 13

• it is the Michaelis-Menten constant, defined as the substrate concentration at half the maximum velocity
• it tells us about the strength of the interaction between the substrate and the enzyme
• it also reflects the specificity that an enzyme shows for a substrate

Question 14

What does a small K_M and a large K_M tell us about the enzyme?

Answer 14

• Small K_M:
• tells us that an enzyme binds strongly to its substrate
• needs a low concentration of substrate the catalyze the formation of the product effectively
• Large K_M:
• enzyme binds weakly with its substrate
• needs a high concentration of substrate to catalyse effectively the formation of the product

Question 15

Describe the Lineweaver-Burk plot

Answer 15

• it determines the K_M and Vmax

Question 16

What is k_cat?

Answer 16

• k_cat is the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration
• in the equation, k₂ = k_cat

Question 17

What does the value of k_cat / K_M show you?

Answer 17

• the value of k_cat / K_M (divided by) is the catalytic efficiency of an enzyme
• it combines an enzyme’s catalytic potential with its ability to bind substrate at low concentration
• therefore, the smaller the K_M and the larger the k_cat, the more efficient the enzyme will be

Question 18

What type of bonds to reversible inhibitors have with enzymes?

Answer 18

• temporary, non-covalent bonds

Question 19

Give the three types of reversible inhibitor and describe how they work

Answer 19

• Competitive Inhibitor:
• the substrate and the inhibitor molecule are similar in shape
• inhibitor binds to the active site of the enzyme
• inhibitor and substrate compete to binds to the active site
• how much inhibitor depends on the concentration of substrate
• addition of sufficient substrate can overcome the effects of competitive inhibition
• Uncompetitive Inhibition:
• the inhibitor does not bind to the unbound enzyme
• it binds once the ES complex has formed
• the release of products is prevented
• addition of extra substrate has no effect on overcoming inhibition
• Non-competitive inhibition:
• the inhibitor binds to a site that is not the active site
• substrate molecules can still bind to the active site
• inhibitor stops enzymes from performing its catalytic function

Question 20

How does a competitive inhibitor affect enzyme kinetics?

Draw out the MM and LB plots

Answer 20

• K_M will increase
• no change in V_max

Question 21

How does an uncompetitive inhibitor affect the enzyme’s kinetics?

Draw MM and LB plots

Answer 21

• V_max and K_M both decrease
• (think about why K_M decreases)

Question 22

Explain using equations below how K_M is reduced with an uncompetitive inhibitor

Answer 22

• the inhibitor binds to the enzyme-substrate (ES) complex to form ESI
• amount of ES complex decrease
• this causes more substrate to bind to the enzyme to maintain the equilibrium
• this increase k₁
• using the derived Michaelis-Menten equation, we see that if k₁ increases, K_M decreases
• a lower concentration of substrate is needed to formed half the maximal concentration of ES, so K_M reduces

Question 23

How does a non-competitive inhibitor affect V_max and K_M? And why?

Draw the MM and LB plots

Answer 23

• V_max decreases
• the inhibitor stops the product from forming
• therefore, the resulting solution essentially becomes a more dilute enzyme solution
• K_M stays the same:
• binding of the substrate is not affected by the inhibitor so K_M is not affected

Question 24

What are cofactors?

Answer 24

• some enzymes require non-protein chemical or metal compounds attached to them to work
• they assist reaction
• usually coenzymes (small, organic, vitamin-derived molecules such as FAD) or inorganic metal ions

Question 25

How does pH affect enzymes?

Answer 25

- the rate of enzyme-catalysed reactions depend on the pH of the solution - each enzyme has an optimal pH

Question 26

How does temperature affect enzyme activity?

Answer 26

- every enzyme has an optimal temperature - generally increasing temperature increase rate of enzyme activity up to its optimum temperature - higher temperatures would denature the enzymes by causing the molecules to vibrate and twist so rapidly that their non-covalent bonds break - if a protein loses its secondary and tertiary structure, it becomes denatured

Question 27

Describe how reversible covalent modification can help regulate enzymes involved in signal transduction

Answer 27

Example: - an enzyme can be phosphorylated and activated/deactivated by another kinase enzyme - this type of regulation is reversible - phosphatase enzymes are able to remove the phosphate group - other common modifying groups are acetyl-, methyl-, carboxyl-

Question 28

Define allosteric inhibitor

Answer 28

- an agent that decrease enzyme activity by binding an allosteric site - allosteric binding causes a conformational change in the enzyme that can result in competitive, non-competitive, or uncompetitive inhibition (or a combination of these effects)

Question 29

Are non-competitive inhibitors the only type of allosteric inhibitors?

Answer 29

- no - all non-competitive inhibitors are allosteric inhibitors but not all allosteric inhibitors are non-competitive

Question 30

How could allosteric binding cause competitive and uncompetitive inhibition effects?

Answer 30

- binding to the allosteric site could cause a conformational change in the active site, preventing substrate binding or obscure/prevent sufficient access to it - causing competitive inhibitory effects - allosteric binding could stabilise an enzyme-substrate complex - resulting in uncompetive inhibition

Question 31

Does allosteric binding always result in inhibition?

Answer 31

- no, it can also result in the activation of an enzyme

Question 32

What does the sigmoid graph of reaction velocity against substrate concentration display?

Answer 32

- most allosterically-regulated enzymes are proteins with a quaternary structure - multisubunit allosteric enzymes show sigmoid shapes - their multiple subunits and their allosteric sites mean that they do not obey Michaelis-Menten kinetics and cannot be differentiated by analysis using Lineweaver-Burk plots

Question 33

Describe the shape of the sigmoid graph

Answer 33

- the reaction gradually increases as substrate concentration increases - after the first active site is filled, other substrates bind are more likely to bind to the other active sites - this is a due to a quaternary change to the enzyme's structure - this increases the reaction velocity - as all the sites are saturated, reaction rate reaches a plateau

Question 34

Why are allosteric enzymes very important in regulating metabolic pathways?

Answer 34

- they are very sensitive to relatively small changes in substrate and also inhibitor concentration