Biochemistry - Ch 11 - Mechanisms Of Enzyme Action

Front 1

Life is based on _____ _____ reactions.

Back 1

...enzyme catalyzed...

Front 2

What are 5 general properties for enzymes?

(i.e. why are enzymes important for life)

Back 2

1) Enzymes increase the reaction rate. (10⁶ - 10¹²)

2) Makes it possible for reaction to take place under *milder conditions*

3) Greater reaction *specificity*

4) Couple reactions (non-spontaneous to spontaneous)

5) Regulation (on or off)

Front 3

What are the 2 types of cofactors?

Back 3

1) Metal ions (inorganic)

2) Coenzymes (organic)

Front 4

What are the 2 types of coenzymes?

Back 4

1) Cosubstrates

2) Prosthetic groups

Front 5

Name 5 metals that may act as metal ion cofactors.

Back 5

{Mn²+, Mg²+, Cu²+, Zn²+, Fe²+}

Front 6

What are 3 examples of Coenzymes?

Which are cosubstrates and which are prosthetic groups?

Back 6

{NAD, CoA, Heme}

Cosubstrate: NAD, CoA(?)

Prosthetic Group: Heme

Front 7

What is the difference between a cosubstrate and a prosthetic group?

Back 7

A cosubstrate is only *transiently* associated with a given enzyme molecule.

A prosthetic group is *permanently* associated with their enzyme.

Front 8

What are apoenzymes and holoenzymes?

Back 8

A holoenzyme is a catalytically active enzyme-cofactor complex.

An apoenzyme is an enzymatically inactive protein resulting from the removal of a holoenzyme's cofactor.

(pg. 327)

Front 9

What's the difference between how prosthetic groups and cosubstrates are regenerated?

Back 9

Prosthetic groups regenerated during the reaction.

Cosubstrates are regenerated in a separate reaction.

Front 10

Enzymes (increase/decrease) the rate of reaction.

Back 10

...increase!

Front 11

What are 3 ways enzymes help to increase the rate of a reaction by binding substrates?

Back 11

1) Immobilize the reactants

2) Orientate the reactants

3) Lower the activation energy.

Front 12

Where does a substrate bind on an enzyme?

Back 12

To the catalytic cleft

(aka Substrate Binding Site)

Front 13

What are 4 characteristics of the catalytic cleft?

(a.k.a. substrate binding site)

Back 13

1) Geometric complementarity (stereospecificity)

2) Electronic complementarity

3) Binding site is usually preformed

4) There can be a conformational change (Induced fit)

Front 14

What is an advantage to enzymes being stereospecific?

Back 14

Chemically identical groups can be distinguished.

Front 15

Describe some ways that the stringency of substrate specificity varies.

Back 15

- Some enzymes are extremely specific (RNAp)

- Other enzymes recognize related molecules.

- Some enzymes have very broad specificity
(e.g. digestive enzymes, lysosomal enzymes)

Front 16

What are 2 examples of enzymes that have a very broad specificity?

Back 16

1) Digestive enzymes

2) Lysosomal enzymes

Front 17

What are the 2 ways we can increase the rate of a reaction?

Back 17

1) Increase the temperature

2) Decrease the energy of the transition state
(e.g. add a catalyst)

Front 18

True/False:

Lowering the energy of the transition state will alter the amount of products &/or you obtain.

Back 18

FALSE.

Changing the energy of the transition state (i.e. making the "activation energy" hump lower) will only increase the rate of the reaction.

You will *not* change the amount of reactants/products you have, just the speed at which you get that amount.

Front 19

Describe how enzymes change the "Reaction Coordinate Diagram" in terms of:

ΔG‡, ΔG⁰, and reaction rate

Back 19

Enzymes:
1) Decrease ΔG‡
- they do *not* change ΔG⁰.

2) Increase the reaction rate
- they do *not* change the equilibrium

Front 20

Name the 6 main catalytic mechanisms for enzymes.

Back 20

1) Acid-Base catalysis

2) Covalent catalysis (nucleophilic)

3) Metal Ion Catalysis

4) Electrostatic catalysis

5) Proximity and Orientation effects

6) Preferential binding of transition state complex

Front 21

What is a reaction that uses both "general acid catalysis" and "general base catalysis" called?

Back 21

A concerted acid-base catalyzed reaction.

Front 22

What are 6 amino acids that usually act in an acid catalyzed or base catalyzed reaction?

Why do these 6 act this way?

Back 22

{Asp, Glu, His, Cys, Tyr, Lys}

These 6 have pK's in or near the physiological pH range which permits them to act as acid and/or base catalysts.

Front 23

Describe the type of enzymic reaction that occurs in Bovine Pancreatic RNase A.

Back 23

It uses concerted acid-base catalysis for hydrolysis of a phosphoester bond

Front 24

What is another name for the enzymic reaction mechanism "Covalent Catalysis"?

Back 24

Nucleophilic catalysis

Front 25

Describe the 3 general steps for Covalent Catalysis.

Back 25

1) Nucleophilic attack by the enzyme

2) Enzyme-substrate intermediate promotes reactivity elsewhere in the substrate.

3) Elimination of the enzyme.

Front 26

What 3 things does the enzyme provide in Covalent Catalysis?

Back 26

1) Fixes the substrate

2) Provides a nucleophile

3) A basic and an acidic group

Front 27

What are the 2 groups of enzymes in Metal Ion Catalysis?

Back 27

1) Metallo-enzymes

2) Metal-activated enzymes

Front 28

What are some example metals of Metallo-enzymes?

What role do they commonly play in Metal Ion Catalysis?

Back 28

Metallo-enzymes:

{Fe²+, Fe³+, Cu²+, Mn²+, Co²+, Mg²+, Zn²+}

These metals play a *catalytic* role in the enzyme (as opposed to structural).

Front 29

What are some example metals of Metal-activated enzymes?

What role do they commonly play in Metal Ion Catalysis?

Back 29

Metal-activated enzymes:

{Na+, K+, Ca²+, Mg²+, Zn²+}

These metals play a *structural* role in the enzyme (as opposed to catalytic).

Front 30

How are the metals:

{Mg²+, Zn²+}

unique in terms of Metal Ion Catalysis?

Back 30

They can play either a structural or a catalytic role in Metal Ion Catalysis.

Front 31

Metal Ion Catalysis:

What are the 3 main ways metal ions participate in catalysis?

Back 31

1) By binding to substrates to orient them properly for reaction.

2) By mediating oxidation-reduction reactions through reversible changes in the metal ions oxidation state.

3) By electrostatically stabilizing or shielding negative charges.

Front 32

What reaction mechanism was used as an example of Metal Ion Catalysis?

Back 32

The role of Zn²+ in carbonic anhydrase.

[11.13] pg. 336

Front 33

What are the 3 main steps in Electrostatic Catalysis?

Back 33

1) Water is usually excluded from the catalytic cleft after substrates are bound.

2) Strong electrostatic interactions

3) Electrostatic interactions stabilize the transition state.

Front 34

What are 2 ways "Proximity and Orientation Effects" act as a catalytic mechanism?

Back 34

1) Bring substrates together

2) Orient substrates so they have the proper orientation

Front 35

How is free energy related to the lowering of the reaction rate in enzyme catalyzed reactions?

Back 35

The free energy released upon binding of a substrate to the enzyme is used to lower the activation energy, supplying functional groups that initiate the reaction.

Front 36

Enzymes mediate ____ of biochemical reactions

Back 36

...coupling...

Front 37

Lysozyme:

Who and when were the 2 people hwo discovered Lysozyme?

Back 37

1909 by P. Laschtchenko in egg white.

1922 by Alexander Flemming ( no pun intended)
- Found that nasal mucus inhibited bacterial growth.
- Proceeded working on this enzyme that he isolated from the tears of volunteers.

Front 38

Lysozyme:

Generally, Lysozyme's main function is to...

Back 38

...cleave bacterial cell walls.

Front 39

Lysozyme:

What is the name of the structure that Lysozyme cleaves in bacterial cell walls?

What is it's general structure?

Back 39

Lysozyme cleaves peptidoglycans.

Peptidoglycans:
1) Linear polysaccharides

2) Crosslinked by short peptides

Front 40

Lysozyme:

Specifically, where does Lysozyme cleave in bacterial cell walls?

Back 40

Lysozyme cleaves the β-glycosidic bond between the NAM1 carbon and the NAG4 carbon

[11.16] pg 340

Front 41

Lysozyme:

Describe the 5 step process by which Lysozyme cleaves bacterial cell walls.

Include the enzymic method by which each step occurs.

Back 41

1. Specificity substrate binding site
– (H-bonds and van der Waals interactions)

2. NAM in the D position is distorted
– (preferential binding of the transition state)

3. Glu 35 donates a proton
– (general acid catalysis)

4. Asp 52 stabilizes the carbocation by forming a
covalent bond
– (covalent catalysis)

5. Glu 35 activates H₂O
– (general base catalysis)

Front 42

Lysozyme:

Describe the substrate binding site of Lysozyme.

Back 42

1) Space for 6 sugars ("a" through "f")

2) C and E do not accommodate NAM

3) NAM must occupy the B, D, and F site

4) NAM in D site has to be distorted.

Front 43

Lysozyme:

What are the 4 catalytic mechanisms used during the Lysozyme reaction?

Back 43

1) Acid catalysis (GLU 35)

2) Preferential binding of transition state

3) Covalent catalysis

4) Base catalysis
- Important for activating base and leaving enzyme as it originally was.

Front 44

Serine Proteases:

What are the 3 Serine Proteases we spoke about?

Back 44

1) Trypsin (+ve charged residues)

2) Chymotrypsin (bulky h-phobic)

3) Elastase (small neutral)

Front 45

Serine Proteases:

Of the 3 we spoke about, what is their main function?

Back 45

They are digestive enzymes that are synthesized by the pancreas and secreted into the duodenum (small intestines).

All these enzymes catalyze the hydrolysis of peptide (amide) bonds but with different specificities for the side chains flanking the scissile (to be cleaved) peptide bond)

Front 46

Serine Proteases:

What are the 2 examples of inhibitors for Chymotrypsin we spoke about?

Back 46

1) DIPF
Diisopropylphosphofluoridate

2) TPCK
Tosyl-L-phenylalanine chloromethylketone

Front 47

Serine Proteases:

How does DIPF inhibit Chymotrypsin?

Back 47

- Irreversibly inactivates chymotrypsin (and other family members)

- Modifies SER 195

- Suggests that SER 195 is important for catalysis.

Front 48

Serine Proteases:

DIPF is a _____ poison.

Back 48

...nerve...

Front 49

Serine Proteases:

What are 3 other nerve poisons with similar chemical compositions to DIPF?

Back 49

1) Malathion

2) Parathion

3) Sarin

Front 50

Serine Proteases:

How does TPCK inhibit Chymotrypsin?

Back 50

- Specific inhibitor of chymotrypsine

- Forms a covalent bond with HIS 57

- Suggests that HIS 58 is involved in catalysis

Front 51

Serine Proteases:

Who are the 3 members of the catalytic triad?

What is their function?

Back 51

• His 57
• Asp 102
• Ser 195

These three invariant residues (within serine proteases) form a hydrogen-bonded constellation referred to as the catalytic triad.

Front 52

Serine Proteases:

Describe the 5 step process for the reaction mechanism of Chymotrypsin.

List the catalytic mechanism that catalyzes each step.

Back 52

[11.29] pg. 353

1) HIS 57 is pulling on the proton from SER 195
- general base catalysis

2) SER 195 forms a covalent bond with the amide carbonyl
- covalent catalysis

3) The oxyanion (transition state) is stabalized by 2 h-bonds
- electrostatic catalysis or preferential binding to the transition state

4) HIS 57 now donates a proton to the transition state, resulting in decomposition of the peptide bond
- general acid catalysis

5) The carboxyl-terminal half of the protein is released
- replaced H₂O and a reversal of the first 4 steps of catalysis

Front 53

Serine Proteases:

Describe the process of how Premature Activation is prevented.

Why is this preventing this a good thing?

Back 53

1) The enzymes are synthesized as Zymogens (proenzymes; i.e. inactive)

2) Pancrease contains inhibitors

3) Storage of zymogens in granules contained within membranes

4) Activators produced by separate cell type.

Because serine proteases are *digestive enzymes*, synthesizing them in their active form would be a bad idea; i.e. the cells/tissues surrounding them would get digested.