Module 5: V5 - V13

Question 1

What is the equilibrium equation for the Michaelis Menten Theory?

Answer 1

E + S ⇌ ES -> E + P

Question 2

What are the assumptions made in the Michaelis Menten Theory?

Answer 2

ES conversion to E + P is irreversible
steady-state conditions [ES] constant (rate of formation of ES = rate of breakdown of ES)
[S]&raquo_space; [Et]
[S]&raquo_space; [P] (initial conditions)

Question 3

What is the equation for the Michaelis Menten Theory?

Answer 3

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

Question 4

What is the equation for the Michaelis constant (Km)?

Answer 4

[E][S]/[ES] = (k2 + k-1)/k1 = Km

Question 5

What are some rules of the Michaelis Menten graph?

Answer 5

when [S]&raquo_space; Km; Vo = Vmax

when [S] = Km; Vo = 1/2 Vmax

Question 6

How is a typical Michaelis Menten plot converted into a linear graph?

Answer 6

by reciprocating both sides of the equation Vo = Vmax [S] / Km + [S]

Question 7

When is Kd taken into account? When is Km taken into account?

Answer 7

under equilibrium conditions

under steady-state conditions (also takes account of the catalytic step)

Question 8

What does a low Km value correspond to?

Answer 8

tighter binding of the enzyme to the substrate

Question 9

What is the turnover number?

Answer 9

k2 which is also the rate constant for the rate-limiting step i.e. the number of molecules converted to product (S to P) per unit time per enzyme molecule saturated with substrate (i.e. when [ES] = [Et]

Question 10

What does a larger turnover number correspond to?

Answer 10

a faster converting enzyme

Question 11

What is the specificity constant?

Answer 11

the rate constant for the conversion of E + S to E + P (kcat/Km)
when [S] &laquo_space;Km, Vo is proportional to (kcat/Km) i.e. kcat/Km reflects both substrate affinity and catalytic efficiency

Question 12

What does a larger value for kcat/Km correspond to?

Answer 12

indicates a more efficient use of the substrate

Question 13

What are irreversible inhibitors of enzymes?

Answer 13

molecules which bind covalently to the active site, destroy a functional group essential for enzyme activity, or form a stable noncovalent complex with the enzyme

Question 14

What are reversible inhibitors of enzymes?

Answer 14

molecules which bind reversibly to enzymes and inhibit the enzyme either by competitive, uncompetitive or mixed modes of inhibition

Question 15

How is a decrease in activity of an enzyme due to competitive inhibition shown in the Michaelis Menten equation?

Answer 15

using an ɑ factor which is placed in the denominator

e.g. Vo = Vmax [S] / ɑ Km + [S]

Question 16

What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without a competitive inhibitor? Why?

Answer 16

the gradient would get steeper as the ɑ factor increases in value
because competitive inhibitors bind to the free enzyme

Question 17

How is a decrease in activity of an enzyme due to uncompetitive inhibition shown in the Michaelis Menten equation? Why?

Answer 17

using an ɑ’ factor which is placed in the denominator
e.g. Vo = Vmax [S] / Km + ɑ’ [S]
because uncompetitive inhibitors bind to the ES complex

Question 18

What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without an uncompetitive inhibitor? Why?

Answer 18

the gradient stays the same, however the y-intercept increases as the ɑ factor increases in value because uncompetitive inhibitors do not bind to the free enzyme and instead bind to the ES complex

Question 19

What is mixed inhibition?

Answer 19

occurs when an inhibitor binds to both the free enzyme and the ES complex

Question 20

How is a decrease in activity of an enzyme due to mixed inhibition shown in the Michaelis Menten equation? Why?

Answer 20

using an ɑ and ɑ’ factor
e.g. Vo= Vmax [S] / ɑKm + ɑ’[S]
because mixed inhibitors bind to the free enzyme and the ES complex

Question 21

What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without a mixed inhibitor? Why?

Answer 21

the gradient and y-intercept both increase as the ɑ factor increases in value because mixed inhibitors bind to the free enzyme and the ES complex

Question 22

What is the role of allosteric enzymes?

Answer 22

often regulate metabolic pathways by changing activity in response to changes in the concentrations of molecules around them

Question 23

What compounds are allosteric enzymes regulated by?

Answer 23

allosteric modulators and allosteric effectors

Question 24

What do positive and negative modulators do?

Answer 24

positive modulators activate and negative modulators inhibit allosteric enzymes

Question 25

How do modulators bind to an allosteric enzyme?

Answer 25

they bind reversibly and non-covalently to the enzyme

Question 26

Which subunits usually exist in an allosteric enzyme and which subunit do modulators bind to?

Answer 26

catalytic and regulatory subunits | modulators bind to the regulatory subunit

Question 27

How does a positive modulator affect an allosteric enzyme?

Answer 27

causes a change in conformation at the active site so the substrate can bind with higher affinity (stabilises the high affinity R state of the enzyme)

Question 28

Do allosteric enzymes show M-M kinetics?

Answer 28

no

Question 29

What happens when S (e.g. for ATCase, S is Asp or carbamyl phosphate) binds?

Answer 29

there is a transition from the T state to the R state to give a sigmoidal Vo versus S plot

Question 30

What process is ATCase involved in and what are positive / negative modulators of ATCase?

Answer 30

ATCase catalyses the first step in the E. coli pathway to produce the nucleotides UTP and CTP CTP is a negative modulator of ATCase when at high levels and ATP is a positive modulator at high levels

Question 31

How many subunits does ATCase have and what type of subunits are they?

Answer 31

ATCase has 12 subunits | 6 catalytic subunits (2 trimeric complexes) and 6 regulatory subunits (3 dimeric complexes)

Question 32

How does CTP and ATP affect the Vo vs [S] plot of ATCase?

Answer 32

as ATP increases, the plot becomes more hyperbola-like (R state) and as CTP increases, the plot shift to the right (T state)

Question 33

What are some structural characteristics of α-chymotrypsin?

Answer 33

5 disulfide bonds within and between the A, B and C chains hydrophobic pocket that binds the side chain of Phe, Tyr or Trp in the substrate protein an active site

Question 34

What is the role of chymotrypsin?

Answer 34

chymotrypsin is one of several proteases that cuts peptides at specific locations on the peptide backbone

Question 35

Which location in a peptide is chymotrypsin able to cleave?

Answer 35

the peptide bond adjacent to aromatic amino acids

Question 36

Why does the sharp increase in activity within chymotrypsin correspond to changes in kcat?

Answer 36

because below pH 7, His57 is protonated and cannot accept the proton from Ser195, so kcat decreases above pH 8, His57 is all deprotonated, so kcat does not change

Question 37

Why does chymotrypsin activity decrease above pH 8.5?

Answer 37

because H+ is lost from the N-ter NH3+ of B chain (Ile16) this results in loss of the Ile16-Asp194 salt bridge which changes the hydrophobic pocket where substrate binds, so 1/Km decreases

Question 38

What does a big value of alpha mean? What does a big value of alpha' mean?

Answer 38

a big value of alpha results in a steep gradient on the double reciprocal plot and a big value of alpha' results in a large y-intercept

Question 39

How is it possible that some allosteric modulators can activate and some can inhibit - the same enzyme?

Answer 39

because modulators can either be positive or negative