Enzymes Flashcards
What are the 2 main components of enzymes?
protein structure - scaffold to provide support
active site - composed of binding and catalytic sites
How do enzymes control metabolism?
The breakage and formation of covalent bonds
What are the only enzymes not composed of proteins?
Riboenzymes
What is an allosteric site?
A site other than the active site on the enzyme
What is a coenzyme?
Small non-protein organic compounds e.g Coenzyme A
Name 4 differences between enzymes and chemical catalysts
- Higher reaction rates
- Work under milder conditions
- Greater reaction specificity
- Capacity for regulation
What do enzymes affect?
Only the kinetics, NO CHANGE IN FREE ENERGY
How can unfavourable reactions be catalysed?
By pairing them with favourable ones
What is the optimal pH for most enzymes?
6-8 (beyond which electrostatic forces break them down)
What are the 6 classifications of enzymes?
1) Oxidoreductases
2) Transferases
3) Hydrolases
4) Lysases
5) Isomerases
6) Ligases
What can act as a cofactor?
- Can be inorganic or organic
- Organic group can be permanently associated with the active site or loosely/reversibly bound
Name 3 ways in which an enzyme can form a complex with a substrate
- Interaction of charged groups
- Formation of H bonds
- Hydrophobic groups fit into enzyme hydrophobic pockets
What is the difference between an apoenzyme and a holoenzyme?
holoenzyme - A catalytically active enzyme-cofactor complex
apoenzyme - The enzymatically inactive protein resulting from removal of the cofactors
What is the two-step model of catalysis
Enzyme works by forming a complex with a substrate and acting on it for a finite period of time
What is the lock and key model?
Where there is 1 active site per molecule which is a perfect fit
What is the induced fit model?
Where the protein changes its structure around the substrate in order to induce a fit
What is the conformational selection model?
Where the protein exists in an equilibrium between different conformers to which one a substrate binds
What does the affinity between between a substrate and enzyme depend upon?
Interaction strength, cooperativity and allostery
What does the affinity refer to?
Strength of the interaction (greater decrease in free energy)
What does specificity refer to?
The relative strength of interactions made between one protein and alternative ligands
How is protein (P) and ligand (L) binding quantified?
In terms of the association (ka) and dissociation (kd) constant. At saturation equilibrium is reached (kA where kA =(P.L)/(P)(L) or 1/k)
Give an equation for change in free energy of binding in terms of dissociation constant
ChangeGbinding = +RT ln kd R= gas constant
How does kD affect change in Gbind
lower kd decreases (negative) Gbind
Define kA and kD
kA = ka/kd kD= kd/ka
How is kD experimentally determined?
Fixed low concentration of receptor and vary the ammount of substrate, measure fraction of bound receptor
In this case the KD is the ligand concentration at which the half of the protein concentration is bound to L
How is KD determined when protein concentration is not directly measurable?
Use statchard analysis (see statchard equation)
slope = -1/KD
intercept = Ptotal/KD
What happens when we try to measure a high affinity system?
- can no longer assume that total ligand concentration and free ligand concentration are the same
- kD no longer represents half occupancy
- Use a more general quadratic equation
What is cooperativity?
Where receptors are multimetric and binding of the first ligand can affect binding of subsequent ligands to other subunits of the complex
What is the Hill equation?
f = [L]n/Kd+[L]n n = Hill coefficient n>1 positive cooperativity n = 1 no cooperativity n <1 negative cooperativity
What are the 5 catalytic strategies?
- Acid-base
- Covalent catalysis
- Metal ion catalysis
- Proximity and orientation effects
- Preferential binding of the transition state
Describe acid-base catalysis
Where the partial proton donation (acid) or
extraction (base) lowers the activation energy and
accelerates the reaction
Describe covalent cataysis
usually a nucleophile group on the enzyme which forms a covalent bond with an electrophile group on the substrate, good cataylsis involves a highly nucleophile species and a good leaving group
How can metal ions act as catalysts?
electrostatically stabilize negative charges formed during Intermediate states of the reaction
By which 4 methods do enzymes catalyze reactions through proximity and orientation effects?
- Bring substrates close to catalytic residues
- Allow binding of substrate in proper orientation
- Stabilization of transition state by electrostatic interactions
- Freezing out translational and rotational mobility of the substrate (up to 10^7 fold)
