Enzyme Kinetics

Question 1

What is enzyme kinetics?

Answer 1

The study of the rate of an enzyme-catalysed reaction and how that rate varies with different conditions

Question 2

What is the rate of a reaction?

Answer 2

Decrease in substrate concentration/increase in product concentration

Per unit time

Question 3

Describe the rate-concentration graph of a typical enzyme with saturation hyperbolic kinetics

Answer 3

Michaelis-Menten

Low substrate concentration = linear/first order as rate is proportional to [substrate]

High substrate concentration= zero order as rate is independent of [substrate]

Question 4

What is Vmax?

Answer 4

The maximum velocity of a reaction that occurs at infinite substrate concentration

Question 5

What is the Michaelis-Menten reaction model?

Answer 5

E + S —> ES —> E + P

With rate constants

Question 6

What assumptions do we make when using the Michaelis-Menten reaction model?

Answer 6

[S] is much greater than [E] so only a small amount of substrate bound at any one time

[ES] does not change with time (formation = breakdown into E+S/P) - steady state approximation

Initial velocities used so [P] is small so E + P —> ES can be ignored (k-2)

Question 7

What equation is used to find the rate of a reaction?

Answer 7

V0 = (Vmax.[S]) / (Km+[S])

Question 8

What does the substrate concentration represent when V0 = Vmax/2?

Answer 8

Km

Question 9

How do you calculate Km using rate constants?

Answer 9

Km = (K2 + K-1) / K1

Question 10

When is Km approximately the Kd for ES?

Answer 10

When K2 is very small/rate-limiting

Question 11

What does Km represent when K2 is rate-limiting?

Answer 11

Measurement of affinity of enzyme for its substrate in the ES complex (Kd)

Question 12

What does a low Km mean?

Answer 12

High affinity of enzyme for substrate

Question 13

What is Kcat?

Answer 13

Turnover number

Number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when saturated with substrate

Question 14

What is the equation relating Vmax, Kcat and [E]t?

Answer 14

Vmax = Kcat.[E]t

Question 15

What does a high Kcat mean?

Answer 15

Reaction can occur faster (higher Vmax)

Question 16

What is the specificity constant?

Answer 16

Number used to quantify catalytic efficiency of enzymes

Question 17

How do you calculate the specificity constant?

Answer 17

Specificity constant = Kcat / Km

Question 18

What does a high specificity constant show?

Answer 18

Enzyme is very efficient

Question 19

What is the Lineweaver-Burk equation?

Answer 19

1/V0 = Km/Vmax . 1/[S] + 1/Vmax

Question 20

What are on the axes of a Michaelis-Menten graph?

Answer 20

X = [S]

Y = initial reaction velocity

Question 21

What are on the axes of a Lineweaver-Burk plot?

Answer 21

X = 1/[S]

Y = 1/V0

Question 22

How do you find 1/Vmax on a Lineweaver-Burk plot?

Answer 22

Y-intercept

Question 23

How do you find -1/Km on a Lineweaver-Burk plot?

Answer 23

X-intercept

Question 24

What does the y-intercept represent on a Lineweaver-Burk plot?

Answer 24

1/Vmax

Question 25

What does the x-intercept represent on a Lineweaver-Burk plot?

Answer 25

-1/Km

Question 26

How can you calculate the gradient of a Lineweaver-Burk plot?

Answer 26

Km/Vmax

Question 27

What is an example of a competitive inhibitor?

Answer 27

Malonate inhibiting succinate dehydrogenase

Question 28

How does a competitive inhibitor work?

Answer 28

Similar chemical structure to substrate Binds to active site of enzyme but does not activate/react

Question 29

What value does a competitive inhibitor affect?

Answer 29

Km = [S] must increase to outcompete inhibitor | Vmax unaffected as inhibitor can be surmounted

Question 30

What value does a non-competitive inhibitor affect?

Answer 30

Vmax = turnover of substrate prevented and effect cannot be surmounted (Km unaffected as inhibitor does not affect affinity of enzyme for its substrate)

Question 31

What do ACE inhibitors do in the body?

Answer 31

Inhibit ACE (angiotensin converting enzyme) So no angiotensin II produced So no periphery vasoconstriction Blood pressure does not increase

Question 32

What is an example of an ACE inhibitor?

Answer 32

Captopril

Question 33

What is a competitive reversible inhibitor of acetylcholine-esterase?

Answer 33

Neostigmine

Question 34

What is a competitive irreversible inhibitor of acetylcholine-esterase?

Answer 34

Diisopropyl fluorophosphate/dyflos

Question 35

How does neostigmine work?

Answer 35

Competitive reversible inhibitor of acetylcholine-esterase Forms covalent bond with Ser (of catalytic triad) Bond is hydrolysed over time

Question 36

How does diisopropyl fluorophosphate work?

Answer 36

Competitive irreversible inhibitor of acetylcholine-esterase Bonds covalently with no spontaneous hydrolysis

Question 37

What can be used to reactivate AChE after using diisopropyl fluorophosphate?

Answer 37

Pralidoxime

Question 38

What are neostigmine and diisopropyl fluorophosphate used to treat?

Answer 38

Alzheimer’s disease

Question 39

How can enzyme activity be regulated?

Answer 39

Allosteric regulators with allosteric enzymes Covalent modification by other enzymes Induction/repression of enzyme synthesis

Question 40

What kind of graph represents the enzyme activity of an allosteric enzyme?

Answer 40

Sigmoid

Question 41

What is an example of allosteric inhibition?

Answer 41

ATP and citrate on phosphofructokinase

Question 42

What is an example of allosteric activation?

Answer 42

Phosphoenol pyruvate (PEP) and fructose-1,6-bisphosphate on pyruvate kinase

Question 43

What residues are usually (de)phosphorylated in covalent modifications?

Answer 43

Ser Thr Tyr His

Question 44

How is covalent modification used to activate and inactivate glycogen phosphorylase?

Answer 44

Phosphorylase kinase phosphorylates Ser 14 using ATP to activate Phosphorylase phosphatase dephosphorylates Ser 14 to inhibit

Question 45

What are some types of covalent modification of enzymes?

Answer 45

Phosphorylation Adenylylation/Uridylylation of Tyr ADP ribosylation (Arg, Gln, Cys) Methylation (Glu) Acetylation (Lys)

Question 46

What happens in adenylylation/uridylylation?

Answer 46

ATP or UDP —> PPi Tyrosine residues

Question 47

What happens in ADP ribosylation?

Answer 47

Arg, Gln, Cys NAD —> nicotinamide

Question 48

What happens in methylation?

Answer 48

Glu S-adenosyl-methionine —> S-adenosyl-homocysteine

Question 49

What happens in acetylation?

Answer 49

Lys on histones Increased transcription

Question 50

What is an example of induction of enzyme synthesis?

Answer 50

High blood glucose stimulates insulin release Insulin increases rate of synthesis of key enzymes involved in glucose metabolism (glucokinase, phosphofructokinase, pyruvate kinase)