Lecture 11 - Enzyme Rate (Michaelis-Menten vs Allosteric Enzymes) Flashcards

1
Q

Enzymes catalyse thermodynamically favourable

reactions by

A

lowering the activation energy.

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2
Q

To model of enzyme catalysis, we use a very simple system in which an

A

enzyme, E, converts a single substrate, S, to a single product, P, that is instantly released.

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3
Q

is the conversion of enzyme, E, converts a single substrate, S, to a single product, P, that is instantly released reversible or irreversible?

A

irreversible

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4
Q

Relative speeds of k1 and k-1 define

A

how tightly

substrate binds.

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5
Q

The rate of catalysis, k2, relates to

A

energy of activation for the transition state.

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6
Q

‘Steady state’ refers to

A

time during which [ES] does not change.

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7
Q

why is ES complex necessary for reaction?

A

so [ES] at any time will govern the rate.

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8
Q

‘progress curve’ measures

A

appearance of product (or disappearance of substrate) with time at steady state.

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9
Q

Following the progress of an enzyme catalysed reaction

we measure…

A

initial reaction velocity (rate) i.e.
near time zero – symbol is V0
(or Vi or Vinit).

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10
Q

What is The effect of enzyme concentration on reaction rate when there is sufficient excess of substrate?

A

amount of enzyme increased, the rate of reaction increases.

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11
Q

when substrate is in excess what is proportional to [E] enzyme concentration?

A

Vo, initial velocity,

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12
Q

As [S], concentration of substrate, is increased, the initial rate V0…

A

increases in a linear way at first.

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13
Q

what does the hyperbolic curve show on V0 vs [S] graph?

A

Enzyme properties.

initial rate (V0) increase linear

Enzyme actives sites are occupied. rate of reaction stops increasing.

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14
Q

what can be identified on a V vs [S] curve?

A

Two kinetic parameters

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15
Q

Vmax

A

maximum velocity possible,

when [S] = ∞.

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16
Q

Km

A

Michaelis constant

substrate concentration at which Vobs = Vmax /2.

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17
Q

The Vobs vs. [S] curve is described by

A

Michaelis-Menten equation:

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18
Q

Michaelis-Menten equation:

A

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

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19
Q

How to determine enzyme kinetic parameters?

A

Michaelis-Menten behaviour

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20
Q

Michaelis-Menten model and assumptions

A
  1. Product is not converted back to substrate.
  2. Haldane’s steady state assumption: the rate of ES
    formation equals the rate of its breakdown; that is

d[ES] / dt = 0

  1. Measuring initial rate ensures [S] does not change
    significantly (and [S] is much greater than [E]).
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21
Q

Michaelis-Menten model and assumptions

what is Haldane’s steady state assumption?
d[ES] / dt = 0

A

Haldane’s steady state assumption: the rate of ES

formation equals the rate of its breakdown;

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22
Q

ES complex converts to E + P with

A

first order kinetics

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23
Q

Single molecule events, like radioactive decay, occur with a set probability, giving

A

first order kinetics.

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24
Q

why will ES ® E + P step follow 1st order kinetics?

A

If each ES complex has the
same chance of going
through the transition state,

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25
Q

When the Michaelis-Menten model fits

Some assumptions:

A
  1. All ES complexes have same rate of reaction.
  2. [S] is in vast excess to [E].
  3. Haldane’s steady state assumption: the rate of ES
    formation equals the rate of its breakdown.
  4. Initial rate is measured. That is, early enough that [S] does not change significantly.
  5. The reverse reaction does not occur.
26
Q

do Cooperative enzymes follow Michaelis-Menten Equation?

A

No

27
Q

what curve does Vobs vs. [S] plot show?

A

Sigmoidal

28
Q

allosteric enzymes examples?

A

Aspartate transcarbamylase (ATCase)

phosphofructokinase

29
Q

Cooperative enzymes do NOT follow Michaelis-Menten Equation.

Vobs vs. [S] plot

A

• Respond more steeply to
intermediate changes in [S].

• Evolve at regulatory points
in metabolic pathways.

• Recall haemoglobin.

30
Q

Allosteric enzymes respond to

A

effectors binding away from the active site.

31
Q

Allosteric enzymes Binding accompanies

A

change of shape,

turn changes enzymatic activity.

32
Q

Allosteric enzymes have

A

multiple subunits and display cooperative behaviour.

33
Q

Both cooperativity and allostery depend on the

A

enzyme switching between active and inactive forms.

34
Q

Allosteric ATCase controls entry

A

to pyrimidine biosynthesis.

35
Q

what is the first ‘committed step’ in making CTP, UTP and TTP?

A

Aspartate transcarbamylase (ATCase)

36
Q

CTP

A

inhibits ATCase

37
Q

ATP (a purine nucleotide)

A

activates ATCase, helping

to balance production.

38
Q

ATCase shows most cooperativity in

A

presence of inhibitors.

39
Q

V0 vs Aspartate plot

what is the curve where ATCase in presence of CTP?

A

Sigmoidal

40
Q

V0 vs Aspartate plot

what is the curve where ATCase in presence of ATP?

A

Hyperbolic

Almost fits Michaelis-Menten model

41
Q

ATCase includes

A

dimer of trimers and trimer of dimers

42
Q

what do Regulatory dimers bind and control orientation of catalytic timers?

A

CTP or ATP

43
Q

Catalytic trimers

A

shift orientation and conformation.

44
Q

Active sites sit at

A

interfaces within trimers.

45
Q

ATCase activation

what state do Top and bottom trimers bind in?

A

T-state,

distorting active site.

46
Q

ATCase activation

what happens when Disengage in R-state?

A

allows substrate binding sites to come closer.

47
Q

what controls glycolysis

A

Phosphofructokinase

48
Q

Phosphorylates fructose-6-phosphate (F6P) to

A

fructose bisphosphate.

49
Q

Phosphofructokinase controls glycolysis.

Inhibited if cell has plenty of

A

ATP, i.e. when glycolysis is not needed for energy.

50
Q

Phosphofructokinase controls glycolysis

Homotetramer is cooperative
when

A

inhibited by ATP or

phosphoenolpyruvate (PEP).

51
Q

Phosphofructokinase conformations

T-state

A

more compact,
stabilised by PEP,
an abundant intermediate of glycolysis.

52
Q

Phosphofructokinase conformations

R-state is stabilized by

A

substrate F6P and ADP.

53
Q

Phosphofructokinase conformations

what swap positions in active site?

A

Arginine 162 and Glutamic acid 161

54
Q

At any given time, some need
to turn on and others need to
drop out.

A
  • Enzyme amount
  • Allosteric control
  • Cell location
  • Proteolytic activation
  • Post-translational modification (e.g. phosphorylation of serine).
55
Q

pancreatic digestive enzymes

A
  • Zymogens are secreted from the pancreas in inactive form.
  • Cleavage by proteases in the gut produces active enzymes.
  • Temporal and spatial control.
56
Q

Zymogens are

A

secreted from the pancreas in inactive form.

57
Q

Michaelis-Menten equation is based on

A

binding theory and simple chemical reaction rates.

58
Q

Derivation depends on

A

assumptions which limit how the equation can be used.

59
Q

Allosteric enzymes change

A

shape and activity.

60
Q

If multimeric,

A

allosteric enzymes often are

cooperative.

61
Q

Allosteric enzymes control

A

metabolic pathways.