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

Question 1

Enzymes catalyse thermodynamically favourable

reactions by

Answer 1

lowering the activation energy.

Question 2

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

Answer 2

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

Question 3

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

Answer 3

irreversible

Question 4

Relative speeds of k1 and k-1 define

Answer 4

how tightly

substrate binds.

Question 5

The rate of catalysis, k2, relates to

Answer 5

energy of activation for the transition state.

Question 6

‘Steady state’ refers to

Answer 6

time during which [ES] does not change.

Question 7

why is ES complex necessary for reaction?

Answer 7

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

Question 8

‘progress curve’ measures

Answer 8

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

Question 9

Following the progress of an enzyme catalysed reaction

we measure…

Answer 9

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

Question 10

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

Answer 10

amount of enzyme increased, the rate of reaction increases.

Question 11

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

Answer 11

Vo, initial velocity,

Question 12

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

Answer 12

increases in a linear way at first.

Question 13

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

Answer 13

Enzyme properties.

initial rate (V0) increase linear

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

Question 14

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

Answer 14

Two kinetic parameters

Question 15

Vmax

Answer 15

maximum velocity possible,

when [S] = ∞.

Question 16

Km

Answer 16

Michaelis constant

substrate concentration at which Vobs = Vmax /2.

Question 17

The Vobs vs. [S] curve is described by

Answer 17

Michaelis-Menten equation:

Question 18

Michaelis-Menten equation:

Answer 18

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

Question 19

How to determine enzyme kinetic parameters?

Answer 19

Michaelis-Menten behaviour

Question 20

Michaelis-Menten model and assumptions

Answer 20

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

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

Question 21

Michaelis-Menten model and assumptions

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

Answer 21

Haldane’s steady state assumption: the rate of ES

formation equals the rate of its breakdown;

Question 22

ES complex converts to E + P with

Answer 22

first order kinetics

Question 23

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

Answer 23

first order kinetics.

Question 24

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

Answer 24

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

Question 25

When the Michaelis-Menten model fits Some assumptions:

Answer 25

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.

Question 26

do Cooperative enzymes follow Michaelis-Menten Equation?

Answer 26

No

Question 27

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

Answer 27

Sigmoidal

Question 28

allosteric enzymes examples?

Answer 28

Aspartate transcarbamylase (ATCase) phosphofructokinase

Question 29

Cooperative enzymes do NOT follow Michaelis-Menten Equation. Vobs vs. [S] plot

Answer 29

• Respond more steeply to intermediate changes in [S]. • Evolve at regulatory points in metabolic pathways. • Recall haemoglobin.

Question 30

Allosteric enzymes respond to

Answer 30

effectors binding away from the active site.

Question 31

Allosteric enzymes Binding accompanies

Answer 31

change of shape, | turn changes enzymatic activity.

Question 32

Allosteric enzymes have

Answer 32

multiple subunits and display cooperative behaviour.

Question 33

Both cooperativity and allostery depend on the

Answer 33

enzyme switching between active and inactive forms.

Question 34

Allosteric ATCase controls entry

Answer 34

to pyrimidine biosynthesis.

Question 35

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

Answer 35

Aspartate transcarbamylase (ATCase)

Question 36

CTP

Answer 36

inhibits ATCase

Question 37

ATP (a purine nucleotide)

Answer 37

activates ATCase, helping | to balance production.

Question 38

ATCase shows most cooperativity in

Answer 38

presence of inhibitors.

Question 39

V0 vs Aspartate plot what is the curve where ATCase in presence of CTP?

Answer 39

Sigmoidal

Question 40

V0 vs Aspartate plot what is the curve where ATCase in presence of ATP?

Answer 40

Hyperbolic Almost fits Michaelis-Menten model

Question 41

ATCase includes

Answer 41

dimer of trimers and trimer of dimers

Question 42

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

Answer 42

CTP or ATP

Question 43

Catalytic trimers

Answer 43

shift orientation and conformation.

Question 44

Active sites sit at

Answer 44

interfaces within trimers.

Question 45

ATCase activation what state do Top and bottom trimers bind in?

Answer 45

T-state, distorting active site.

Question 46

ATCase activation what happens when Disengage in R-state?

Answer 46

allows substrate binding sites to come closer.

Question 47

what controls glycolysis

Answer 47

Phosphofructokinase

Question 48

Phosphorylates fructose-6-phosphate (F6P) to

Answer 48

fructose bisphosphate.

Question 49

Phosphofructokinase controls glycolysis. Inhibited if cell has plenty of

Answer 49

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

Question 50

Phosphofructokinase controls glycolysis Homotetramer is cooperative when

Answer 50

inhibited by ATP or | phosphoenolpyruvate (PEP).

Question 51

Phosphofructokinase conformations T-state

Answer 51

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

Question 52

Phosphofructokinase conformations R-state is stabilized by

Answer 52

substrate F6P and ADP.

Question 53

Phosphofructokinase conformations what swap positions in active site?

Answer 53

Arginine 162 and Glutamic acid 161

Question 54

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

Answer 54

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

Question 55

pancreatic digestive enzymes

Answer 55

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

Question 56

Zymogens are

Answer 56

secreted from the pancreas in inactive form.

Question 57

Michaelis-Menten equation is based on

Answer 57

binding theory and simple chemical reaction rates.

Question 58

Derivation depends on

Answer 58

assumptions which limit how the equation can be used.

Question 59

Allosteric enzymes change

Answer 59

shape and activity.

Question 60

If multimeric,

Answer 60

allosteric enzymes often are | cooperative.

Question 61

Allosteric enzymes control

Answer 61

metabolic pathways.