Lecture 9 & 10, enzymatics Flashcards
What is a common catalytic domain in enzymes (specifically serine proteases)?
- Describe:
- Convergent evolution?
Catalytic triad proteases S H D Or D H S - Describe: OH from Ser interacts with one N from His, O- on Asp interacts with the N - H on His. Stabilizes the pocket. - Convergent evolution? This has evolved in multiple different protein backgrounds. Many enzymes have different fold converging into the same catalytic domains.
Kinetics involves consideration of what three things?
Distance (bond distance for example)
Energy
Time (such as rates)
Keq for A + B -> C + D?
- Units?
keq = ([C] [D} / [A] [B]) keq = k(forward) / K(reverse) - Units? Depends. For 2A + B -> C + D K units of M ^ -1 A + B -> 2C + 2D K units of M ^ 2 etc.
Arrhenius Equation:
K = A * e ^ (-ΔG# / (R * T))
K = forward or reverse reaction rate
A = constant which describes how often molecules collide in the correct orientation when all reactants are at 1 mol per liter (1 molar)
e is e in math
ΔG# = the energy of activation
R = universal gas constant, 8.314 J / mol. K
T = temp in kelvin
Are enzymes complementary for the substrate, the transition state, or the product?
Why?
Transition state
- Why?
1: If the substrate was stabilized, its free energy is reduced (DeltaG), it must over come an even larger activation energy then to become the product.
2: Product wouldn’t effect the reaction, though it might stop the enzyme from unbinding.
3: Being complementary to the transition state lowers the deltaG during the period when its highest (the activation energy), this is what needed to become smaller
ΔG = ?
ΔH - TΔS
desolvation:
the process of dissociating or releasing water molecules electrostatistically bound to a particle in aqueous solution.
Ground state destabilization:
- Define it:
- How is it counterbalanced?
- Define it:
Loss of entropy due to forming specific enzyme substrate complex. This means you have made the substrate less stable. Since it is less stable, it will cost less energy to stabilize it (you’ve essentially moved it closer towards the top of the activation energy cost, but in a single small step) - How is it counterbalanced?
Desolvation when the substrate adheres frees water, which lowers entropy cost to water. May counter balance the decrease in entropy caused by substrate enzyme complex formation.
When you form an enzyme substrate complex, you often increase electrostatic interaction. How?
You have moved the interaction into the more hydrophobic protein, lowered the dielectric, increased the electrostatic interaction strength.
Near attack geometry:
- Other name:
Theory that enzymes work by moving a nucleophile near to the electrophile. By holding them near each other, and giving them the right orientation, you increase the odds of the reaction occurring.
- Other name:
Catalysis by approximation
Enthalpy entropy balance in protein surface:
The surface of protein has more loose loops, which allows them to have many interactions with water, and have a high entropy (they can move wherever).
This entropy of the surface helps to counteract the entropy loss of binding the substrate.
While the surface is soft and moves, interacting with water the core is rigid and more constant.
Name five fundamentals of enzyme catalysis:
1: Transition state complementarity (explains why the enzyme must be complementary to the substrates transition state)
2: Ground State Destabilization (loss of entropy when substrate binds, destabilizes it, makes it easier to react into the product state)
3: Increased electrostatics interaction due to desolvation (you’ve placed ions in a more hydrophobic pocket, they bond more strongly in the catalytic site)
4: Near attack conformation (groups which need to attack each other are positioned near each other in enzyme substrate complex)
5: Induced fit (the enzyme is designed to be more relaxed when it is placing the substrate in the ideal place to react, allowing the enzyme to relax lowers activation energy cost.
General Acid/Base catalysis:
Catalysis in which a molecule (other than water) acts as a proton donor or acceptor in the reaction. Initial protonation states are formed in the end.
- Essentially, they help to stabilize the charge as it is moved.
Covalent catalysis:
Enzyme substrate complex formed with hydroxyl or thiol side chains (Cys or Ser commonly)
Oxidoreductase:
- Define:
- Other names:
- Define:
Shifting of electrons around (something is oxidized, something else is reduced) - Other names:
Dehydrogenase, oxidase, monooxygenyase, hydroxylase, reductase
Transferase:
- Define:
- Other names:
- Define:
Mediate transfer of a group from one enzyme to another (like a phosphate, methyl or ubiquitin) - Other names:
kinase, transaminase, transmethylase
Hydrolases:
- Define:
- Other names:
- Define:
mediate breaking of bonds by addition of water, (or formation of bonds by removal of water, but not as common) - Other names:
phosphatase, peptidase, esterase, lipase
What system uses a lot of redox reactions?
Metabolism
Why is NADH useful for studies in enzymes?
It has resonance rings, and when it becomes NAD+ it gains resonance, which means you can detect this difference in UV.
NADH is detected at higher UV wavelength.
What are two other groups with different UV spectrum absorptions depending on if they are oxidized or reduced?
Flavin prosthetic group
Iron porphyrin
How can you tell the oxidation state of iron by UV light?
Fe2+ has more electron then Fe3+, will result in it being larger, pushes the perforin to expand, gives it a slightly different UV absorption pattern
Substrate reactivity increasing with delocalization of _______.
How does this relate to chemical groups?
Electrons
Also called electron dispersion
thiolester > Ester > Amide
Draw an ester reacting with water in hydrolysis.
YAY!
Reactions with chromophoric substrate:
A reaction product is generated which is detectable by UV radiation
You can attack serine to tag it with a chromophoric substrate.
What reacts with what?
How would you figure out what molecule you have?
- What reacts with what?
OH of serine attacks the sulfur. Proton is lost from serine. The F makes like a leaving group and leaves. - How would you figure out what molecule you have?
Run it through mass spec, figure out what is attached to the tag.
If you continue to run enzymes in an in vitro system, what usually happens to them?
The enzyme eventually starts to slow, it loses its cofactor, its catalysis is not as smooth once it has turned over too many times.
If enzymes degrade when you run them too much how do you get a curve of velocity vs substrate conc. ?
You measure initial reaction rate with [S] > > [E], and only measure for a short time. Then you repeat this with less and less enzyme, and use all these points to make a curve (likely logarithmic or sigmoidal)
- Have to keep pH constant
- Have to keep temperature constant
Is E + S - > ES or ES -> E + P the rate limiting step?
ES -> E + P
So this is kcat
V = Vmax [S] / ([S]+ Km)
V = Vmax [S] / ([S]+ Km)
Km = ?
Km = Breakdown rate / formation rate
Km = (k-1 + kcat) / k1
Km is in units of Molarity (the conc. of the substrate will be
What is Km?
The concentration of substrate at which the enzyme will run at half its maximal rate.
What does Km tell you?
With how much affinity the enzyme binds and catalyzes the substrate. A low Km means it is very specific for that site.
High Km suggests weak substrate binding.
Catalytic triad:
Many catalytic sites will have a Acid, Base and Nucleophile (I.E. Asp, His, Ser).
They will also have an oxyanion hole. (but this isn’t part of the triad
Acid: proton donor (I.E. Asp, Glu, His)
Base: Proton acceptor (I.E. His, Ser, lysine)
Nucleophile: often will be deprotonated by (I.E.
How will cleavage by catalytic triad normally work?
Asp stabilizes a H on His, His takes the H from serine. Ser, now O- makes an attack, usually at an ester, thiolester and sometimes at an amide
oxyanion hole:
Stabilizes the anionic intermediate. Usually involves a basic amino acid, an amide or the NH from the backbones of Ser and perhaps a glycine.
