Enzyme Catalysis Flashcards
What is the mass range for enzymes?
10 kDa –> 1000 kDa
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
What are cofactors?
Small non-protein molecules that bind to many enzymes and facilitate their catalytic activity
Holoenzyme
with cofactor
ACTIVE
Apoenzyme
Without cofactor
INACTIVE
Inorganic cofactors
Metal Ions
Examples:
1) Carbonic anhydrase- Zn2+
2) Hexokinase- Mg2+
Organic cofactors
Derived from vitamins
Called coenzymes
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)
What is the job for proteases?
Hydrolyzes peptide bonds
-also hydrolyzes closely related ester bonds
Papin
Cleaves any peptide bond
Trypsin
Splitting peptide bonds only on the carboxyl side of lysine and arginine residues
Thrombin
very specific
Hydrolyzes arginine-glycine bonds in particular peptide sequences
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
ΔG
Spontaneous Exergonic
ΔG > 0
Not spontaneous endergonic
ΔG = 0
Equilibrium
Equilibrium conditions
ΔG= 0
ΔG˚’= -RTlnK’eq
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
ΔG formula
ΔG= ΔG˚’ + RTln [products]/[reactants]
ΔG˚’ 1
Products favored
ΔG˚’ > 0
K’eq
Reactants favored
Can enzymes change the equilibrium of a reaction?
Nope
Equilibrium only depends on difference in free energy between products and reactants
Transition State (X)
Has a higher free energy and lower stability than either S or P
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
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
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+
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
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
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)
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
What does the formation of many reversible noncovalent interactions do?
Releases free energy (binding energy) b/w substrate and the enzyme
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
When is maximum binding energy released?
When the enzyme facilitates the formation of the transition state
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
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
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
Genera acid-base catalysis
Molecule other than water becomes a proton donor or acceptor
I.e.: histidine residue in the active site of chymotrypsin
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
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
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
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
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
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
Oxyanion [Hole]
Provides for hydrogen bonding with the extremely negative oxygen
Creates binding energy in the tetrahedral intermediate
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
