Enzyme Kinetics

Question 1

Speed-up chemical reactions in cells by…

Answer 1

placing the substrate in an environment
that facilitates the reaction

Question 2

Enzymes are proteins with…

Answer 2

catalytic activity

– A catalyst increases the rate of a chemical reaction without being consumed

– 103
-1020 x faster than uncatalyzed reactions

– Exhibit stereospecificity

– Function under physiological conditions

Question 3

What is Enzyme Kinetics?

Answer 3

• Study of the rates, mechanisms and
  properties of enzyme-catalyzed
  reactions
• Provides information on enzyme
  specificities, regulation and adaptation
• Clinically important for diagnosis based on changes in enzyme activity or
  amount

Question 4

Enzymes are very powerful

Answer 4

Catalysis

Question 5

Enzymes can distinguish between…

Answer 5

enantiomers

Question 6

Some enzymes require
additional…

Answer 6

chemicals or
groups to function:

– Metal ions

– Prosthetic groups
* Heme

– Co-factors
* ATP

Question 7

6 types of chemical reactions are catalyzed by enzymes:

Answer 7

– 1. Oxido-reduction: oxidoreductases

– 2. Transfer of chemical groups: transferases

– 3. Hydrolysis: hydrolases

– 4. Removal of chemical groups: lyases

– 5. Isomerisation: isomerases

– 6. Linking two groups together: ligases

Question 8

Oxidoreductases:

Answer 8

Example: Lactate dehydrogenase
(EC 1.1.1.27)

• Lactate = substrate
• Pyruvate = product
• NAD+
  /NADH = co-factor

Question 9

2. Transferases:

Answer 9

Example: Alanine transaminase
(EC 2.6.1.2)

• L-alanine / a-ketoglutarate = substrates
• Pyruvate / L-glutamate = products

Question 10

3. Hydrolases:

Answer 10

Example: diphosphate phosphohydrolase(EC 3.6.1.1)

• Pyrophosphate / H2O = substrates
• Phosphate = product

Question 11

4. Lyases (aka synthases):

Answer 11

Example: Pyruvate decarboxylase(EC 4.1.1.1)

• Pyruvate = substrate
• Acetaldehyde and CO2 = products

Question 12

5. Isomerases:

Answer 12

Example: Alanine racemase
(EC 5.1.1.1)

• L-alanine = substrate
• D-alanine = product

Question 13

6. Ligases (aka synthetases):

Answer 13

Example: L-glutamine synthetase(EC 6.3.1.2)

• L-glutamate / NH4
  +
  = substrates
• L-glutamine = product
• ATP = co-factor

Question 14

Enzymatic reactions take place in…

Answer 14

multiple steps
involving reaction intermediates.

• While in theory these reactions are reversible, in practice, the low levels of the one of the reactants (S or P) usually pushes the equilibrium in one direction.

Question 15

Enzymes increase the rate of …

Answer 15

chemical reactions, but do NOT alter the direction of the equilibrium.

Question 16

In chemical reactions, three conditions
must be met for a reaction to take place:

Answer 16

1. the molecules must collide to react
2. there must be enough energy
   (energy of activation) for the two
   molecules to react.
3. the molecules must be orientated
   correctly with respect to each other.

Question 17

1. the molecules must collide to react.

Answer 17

If two molecules simply collide, however,
they will not always react; therefore, the
occurrence of a collision is not enough.

Question 18

2. there must be enough energy
   (energy of activation) for the two
   molecules to react.

Answer 18

If two slow molecules
collide, they might bounce off one another
because they do not contain enough
energy to reach the energy of activation
and overcome the transition state (the
highest energy point).

Question 19

The transition state is not only a reaction
intermediate:

Answer 19

it is a transitory molecular
structure that is no longer the substrate, but
not yet the product.

Question 20

In the absence of an enzyme, the substrate requires

Answer 20

a
substantial amount of energy in order to reach the activation state and react

Question 21

In the presence of an enzyme, the reaction is facilitated because…

Answer 21

the enzyme provides a better environment for the reaction to occur:

– Close proximity of substrate and chemical groups of the enzyme

– Proper orientation of the chemical groups with respect to the substrate

– The formation of the transition state is favoured

Question 22

The « lock and key » thing is a…

Answer 22

a MYTH:

– If the enzyme and substrate were perfectly
complementary, like a lock and key, the interaction between E and S would be so stable that the
reaction would not occur!

• Instead, the 3D shape of the enzyme is complementary to the
  transition state;
• By doing so, the enzyme favours the formation of the transition
  state, lowers the energy of activation, and accelerates the
  reaction…COOL!

Question 23

Factors influencing enzyme activity

Answer 23

1. Temperature
2. pH
3. Substrate concentration
4. Product concentration
5. Presence of inhibitors (or activators)

Question 24

1. Temperature Factor:

Answer 24

Reflects weak interactions holding
native conformation together

Question 25

2. pH Factor

Answer 25

Due to presence of charged amino acids in the active site

Question 26

How do we measure enzyme activity?

Answer 26

1. Detection of the product(s): – pNA = para-nitroaniline  Absorbs at 405 nm 2. Accumulation/utilisation of a co-factor: – NADH = absorbs strongly at 340 nm (e = 6.3 mol.L-1cm-1) – NAD+ =does not absorb at 340 nm 3. Coupled reactions: – Very useful when neither substrate/product/co-factor can be (easily) detected directly

Question 27

v0 (initial velocity) is the...

Answer 27

rate of the reaction very early on when [P] is negligible

Question 28

v0 can be obtained by...

Answer 28

taking the slope of the graph of [P] vs Time (units: concentration / time)

Question 29

v0 varies as a

Answer 29

function of [E]

Question 30

v0 increases as a function of...

Answer 30

[S] until E is saturated by S

Question 31

When E is saturated with...

Answer 31

S --> v0 = Vmax

Question 32

Michaelis-Menten Equation:

Answer 32

The relationship between vo and [S] can be viewed as a 2 step reaction. This relationship can be expressed by the MichaelisMenten equation.

Question 33

Michaelis-Menten Equation (Km):

Answer 33

Km can be calculated as the [S] required to acheive half the Vmax * Km is a measure of the affinity of E for S – The lower the Km, the less S is requried by E to acheive ½ Vmax, and the greater the affinity of E for S.

Question 34

Turnover number is the...

Answer 34

maximum number of substrate molecules converted to product per second by each active site (units = s-1) The inverse is the amount of time required for E to convert 1 substrate molecule to the product (i.e. time for 1 catalytic event). Units: s.

Question 35

Neither Km nor Vmax can be easily obtained ...

Answer 35

directly from kinetic data because Vmax is rarely acheived (its an hyperbolic curve…)

Question 36

Michaelis-Menten equation describes the relationship between...

Answer 36

reaction rate and substrate concentration. It can explain the saturation behavior in catalyzed reactions as shown in the previous slide.

Question 37

Michaelis-Menten equation is derived based on the following three conditions:

Answer 37

1) State steady assumption; 2) Initial velocity assumption; 3) Rate law.

Question 38

When enzyme is mixed with a high concentration of substrate [S], the...

Answer 38

reaction goes rapidly to steady state. – Does not allow characterization

Question 39

Vmax is asymptotically approached as...

Answer 39

substrate is increased, but never reached in reality, because this requires that ALL enzymes are bound with substrate (think about chemical equilibrium);

Question 40

Two main types of inhibition exist:

Answer 40

– Reversible enzyme inhibition: enzyme activity can be recovered by removing the inhibitor (e.g. dialysis, gel filtration) – Irreversible enzyme inhibition: inhibitor binds covalently to enzyme, which is then irreversibly inactivated

Question 41

VEGF (Vascular Endothelial Growth Factor):

Answer 41

Example of enzyme inhibition. – Produced in embryos and tumours; – Acts via a cell surface receptor to trigger the growth of blood vessels; – Why inhibit VEGF-R: * Blocking the action of VEGF (an enzyme) will block the growth of blood vessels and starve tumours to death!

Question 42

Sildenafil:

Answer 42

Example of enzyme inhibition – cGMP-Phosphodiesterase inhibitor; – Initially tested as an anti-hypertension drug -

Question 43

1- Competitive inhibition

Answer 43

* Most frequently encountered inhibitors; * I is very similar to S – (i.e. it is a structural analog) * I and S compete for the same binding site on the enzyme: the active site; * Vmax stays the same: – At high enough [S], S will outcompete I * Km is increased (Km app): – Because I can bind E, the amount of S required to reach ½ Vmax will be increased.

Question 44

2- Uncompetitive inhibition

Answer 44

* I only bind to ES, not the free enzyme; * Example: glyphosate (Round-up herbicide) * Vmax is decreased: – Some of the E is converted into an inactive ESI complex. * Km is decreased: – I reduces the amount of E that can participate in the reaction; – ESI shifts the E + S ES to the right, leading to an apparent decrease in Km.

Question 45

3- Noncompetitive inhibition:

Answer 45

* I and S bind to different sites on E; * Binding of I on E does not affect the binding of S on E (and vice versa); * So: Km is unchanged, but Vmax is decreased (I reduces the [E] that can generate P); * e.g. deoxycyclin (an antibiotic), that inhibits collagenase (a proteolytic enzyme involved in periodontal diseases).

Question 46

irreversible inhibitors bind covalently to the...

Answer 46

enzyme and permanently inhibit it.

Question 47

Irreversible enzyme inhibition types:

Answer 47

– Group-specific – Active site-directed reagents (aka Affinity labels) – Suicide inhibitors

Question 48

Three main types of catalysis are found:

Answer 48

General acid/base catalysis: Metal ion catalysis: Covalent catalysis:

Question 49

General acid/base catalysis:

Answer 49

Involves the transfer of protons between side chains of the enzyme and the substrate; The most common form of enzyme catalysis

Question 50

Metal ion catalysis:

Answer 50

Some enzymes use metal ions for catalysis (Mg+2 , Mn+2, Zn+2, Fe+2, Cu+2) The metal ion can act is different ways: Stabilise transition state Help orientate the substrate vs enzyme Participate in transfer of electrons/protons between enzyme and substrate

Question 51

Covalent catalysis:

Answer 51

Involves the formation of a transientcovalent bond between the enzyme and substrate; Covalent bond activates substrate for further reaction

Question 52

In any organism, enzymes are always kept in check so that their activity can match the needs of the cell:

Answer 52

– Enzymes can be activated: the reaction is stimulated to generate more product; – Enzymes can be inactivated (or inhibited): the reaction is slowed down to decrease the amount of product;

Question 53

* Several strategies are used to modulate (i.e. activate or inhibit) enzymes:

Answer 53

– 1. Allostery * Inhibition by product * Activation by substrate/cofactor – 2. Binding of regulatory subunits – 3. Covalent modification* Phosphorylation on Ser/Thr/Tyr – 4. Degradation of the enzyme – 5. Limited proteolysis–

Question 54

1. Allostery

Answer 54

binding of an effector molecule to a separate site on the enzyme

Question 55

Allostery is widely used by

Answer 55

metabolic enzymes: – Inhibition by the end product of a pathway; – Activation by a product generated early on in the pathway.

Question 56

(Allostery) Based on the principle of cooperativity:

Answer 56

– The binding of a small molecule to the enzyme modifies the 3-D structure of the protein and altersits ability to catalyse the reaction.

Question 57

2. Regulatory subunits

Answer 57

* cAMP is produced from ATP by the action of adenylate cyclase. * The binding of cAMP to the regulatory subunits of PKA frees the catalytic subunits, that are then fully active.

Question 58

3. Regulation by covalent modification:

Answer 58

* Specific amino acid side chains of several enzymes are the target of covalent modifications (catalysed by yet other enzymes…).

Question 59

Regulation by phosphorylation: Addition of a phosphate group (phosphorylation) by

Answer 59

protein kinases or their removal by protein phosphatases is frequently used to modulate the activity of enzymes

Question 60

4. Regulation of enzyme stability:

Answer 60

* Proteins are constantly being made (synthesized) and destroyed (degraded). * The tight regulation of protein synthesis and degradation is a key factor in the regulation of enzyme activity.

Question 61

4. Regulation of enzyme stability (Ubiquitin:)

Answer 61

– 76 amino acid protein – Tags other proteins for degradation * Ubiquitin is attached to other proteins by a series of 3 enzymes (E1, E2 and E3)

Question 62

4. Regulation of enzyme stability: Polyubiquitylated proteins

Answer 62

are targeted to a HUGE protein complex called the proteasome: – Contains several subunits with different types of protease (i.e. protein hydrolases) activities.

Question 63

Degradation of an enzyme results in a decrease in the...

Answer 63

amount of its product that can be made.

Question 64

5. Limited proteolysis:

Answer 64

* Several enzymes (particularly digestive enzymes) are initially synthesized as inactive precursors (called zymogens / proenzymes). * Activation of the enzymes is done by the cleavage of a limited number of peptide bonds (usually 2-3); * The mature enzyme is made up of 2 to 3 chains, held together by disulfide bonds

Question 65

5. Limited proteolysis: ( Pancreatic trypsin inhibitor)

Answer 65

- inhibits trypsin, and prevents trace amounts of activated trypsin from triggering the proteolytic cascade in the pancreas/pancreatic ducts; * Inherited deficiencies in a similar protease inhibitor (a1-antitrypsin, which inhibits elastase), leads to damage to the lungs and emphysema.