Enzyme Catalysis Flashcards Preview

MoMF > Enzyme Catalysis > Flashcards

Flashcards in Enzyme Catalysis Deck (45):
1

What is the mass range for enzymes?

10 kDa --> 1000 kDa

2

How fast do enzymes accelerate reactions?

They accelerate reactions by a factor of 10^6 or more

I.e.:
1) Carbonic anhydrase 7.7 x 10^6
2) Triose phosphate Isomerase 1.0 x 10^9
3) OMP decarboxylase 1.4 x 10^7

3

What are cofactors?

Small non-protein molecules that bind to many enzymes and facilitate their catalytic activity

4

Holoenzyme

with cofactor

ACTIVE

5

Apoenzyme

Without cofactor

INACTIVE

6

Inorganic cofactors

Metal Ions

Examples:

1) Carbonic anhydrase- Zn2+
2) Hexokinase- Mg2+

7

Organic cofactors

Derived from vitamins
Called coenzymes

8

What are the two types of coenzymes?

1)Co-substrate
-loosely bound
-changed by the reaction
-i.e.: lactate dehydrogenase NAD+ (from niacin)
-can be used by an enzyme and then re used

2) Prosthetic group
-Tightly or covalent lay bound
-not changed by the reaction
-monoamine oxidase FAD (from riboflavin)

9

What is the job for proteases?

Hydrolyzes peptide bonds
-also hydrolyzes closely related ester bonds

10

Papin

Cleaves any peptide bond

11

Trypsin

Splitting peptide bonds only on the carboxyl side of lysine and arginine residues

12

Thrombin

*very specific*

Hydrolyzes arginine-glycine bonds in particular peptide sequences

13

Free energy change

DeltaG= Gp- Gr

*independent of the path that is followed in converting reactants to products

*only considers initial and final states

*give no info about rate/kinetics of reaction

*gives info about spontaneity of reaction

14

ΔG

Spontaneous Exergonic

15

ΔG > 0

Not spontaneous endergonic

16

ΔG = 0

Equilibrium

17

Equilibrium conditions

ΔG= 0

ΔG˚'= -RTlnK'eq

18

ΔG˚'

Standard free energy change for given reaction at standard conditions of:

P=1 atm
[X]= 1 M
PH= 7

No actual standard temperature, ΔG˚' just determined at the temp of the system

19

ΔG formula

ΔG= ΔG˚' + RTln [products]/[reactants]

20

ΔG˚' 1

Products favored

21

ΔG˚' > 0

K'eq

Reactants favored

22

Can enzymes change the equilibrium of a reaction?

Nope
Equilibrium only depends on difference in free energy between products and reactants

23

Transition State (X)

Has a higher free energy and lower stability than either S or P

24

ΔG+

Free energy of activation
Enzymes accelerate reactions by lowering ΔG+ and facilitating the formation of X (transition state)

A relatively small decrease in ΔG+ --> greater increase in the reaction rate

25

What does a 20% decrease in ΔG+ do?

20% decrease in ΔG+ can increase the reaction by 10 fold

*80% decrease --> 10000 fold increase in v

26

Active Site of Enzymes

Region where binds to substrates and other cofactors

Contains catalytic groups (2-3 residues) --> directly participate in making and breaking bonds

Interaction of enzyme and substrate at active site promotes formation of transition state

Active site most responsible for lowering ΔG+

27

Active Site structure

Occupies a 3D cleft or crevice with special micro-environment

Water usually excluded from cleft

Takes up small % of total enzyme volume

28

How does the active site bind substrates?

Weak interactions
-electrostatic
-van Der Waals
-H bonding

Controls specificity of binding through precise orientation of atoms in the site

29

Active site of Lysozyme

Contains 129 AA

Only 6 important residues (5% of total sequence)

Only 2 are catalytic groups (2% of total sequence) (Glutamate and Aspartate)

30

Active site interactions

substrates found to the active site through multiple noncovalent interactions
^only become significant when numerous substrate atoms come close to numerous enzyme atoms

31

What does the formation of many reversible noncovalent interactions do?

Releases free energy (binding energy) b/w substrate and the enzyme

32

What is the binding energy?

Represents the lowering of the activation energy by the enzyme

It is released by the formation of many weak interactions from the induced fit

33

When is maximum binding energy released?

When the enzyme facilitates the formation of the transition state

34

Lock and key model

Unbound enzyme has a rigid active site

Substrate has a shape complementary to the active site

Enzyme and substrate fit exactly into each other

Model explains enzyme specificity but not the stabilization of the transition state by the enzyme

35

Induced fit model

Unbound enzyme has flexibly active site
Substrate has arbitrary shape relative to active site

After substrate binds, enzyme changes shape and become complementary to the substrate shape

Explains both specific you and stabilization and release of binding energy

*substrate and enzyme both change and modify to each other*

36

Covalent catalysis

Active site contains a reactive group (usually powerful nucleophile) that makes a temporary covalent attachment to the substrate during the reaction

I.e.: chymotrypsin active site peptide hydrolysis --> O in serine attacks carbonyl C on peptide bond to form a covalent bond

37

Genera acid-base catalysis

Molecule other than water becomes a proton donor or acceptor

I.e.: histidine residue in the active site of chymotrypsin

38

Catalysis by approximation

An enzyme brings together two distinct substrates along a common binding surface

I.e.: carbonic anhydrase binding CO2 and water in adjacent sites to facilitate their reaction

39

Metal Ion Catalysis

1) promote the formation of nucleophiles by direct coordination (during reaction)

I.e.: Zn for carbonic anhydrase in CO2 hydration

2) act as electrophile said by stabilizing a negative charge on a reaction intermediate (after reaction)

I.e.: Mg for EcoRV endonuclease in DNA hydrolysis

3) serve as a bridge between enzyme and substrate

I.e.: Mg for myosin in ATP hydrolysis

40

Chymotrypsin

A protease

Cleaves C terminal aromatic and methionine (large hydrophobic)

Use of catalytic triad- Ser195, His57, Asp102 (only 1.2% of total active site AA)

Made up of 3 chains= 241 residues

41

Basics on ln (math)

Ln of decimal is negative

As fraction becomes smaller, the ln term becomes even more negative

Ln0.5= -0.69
Ln0.18= -1.78

42

Peptide hydrolysis- favorable conditions

It is thermodynamically favorable but kinetic ally unfavorable b/c of the partial double bond character on the peptide bond -->harder to cleave due to the slight + on carbon
--> also not a very good electrophile so the nucleophile can't come in and attack properly

43

General idea of catalytic triad- Stage 1

Acylation- formation of covalent Acyl-enzyme intermediate

1) His accepts proton from OH group of serine --> Gen base catalysis

2) Alkoxide ion (O-) formed on serine after proton transferred '

3) O- attacks and forms a covalent bond with the peptide bond that is "hard to cleave" --> specifically attacks the carbon --> formation of tetrahedral intermediate

4) Asp is providing stabilization to the amide group in His

5) tetrahedral intermediate creates an oxyanion hole due to negative charge on the once double bonded oxygen on the substrate

6) His proton (originally from serine) bonded to the N on the once peptide bond

7) cleavage of N with proton (reformation of C=O) and free peptide formed with H bonding with N on His

44

Oxyanion [Hole]

Provides for hydrogen bonding with the extremely negative oxygen

Creates binding energy in the tetrahedral intermediate

46

General idea of catalytic triad- Stage 2

Deacylation- regeneration of free enzyme

1) A water H bonds with N on His, leading to a negative O in the water and a good nucleophile. This is the binding energy in the reaction

2) the O- attacks C=O bond

3) formation of Oxyanion again with the loss of the double bond becoming a C-O