1 - Thermodynamics, Enzymes, Inhibitors (Biochemistry I)

3 conditions for V vs. [S] graph:

- [S] << [E] (enzyme in excess)
- [S] ~ [E]
- [S] >> [E] (Vmax reached)

What does Vmax depend on?

- [E]
- Specific enzyme being used

Kinetics:

relating to rates, mechanism, catalyst, intermediate, transition state, activation energy, etc.

Thermodynamics:

relating to stability, equilibrium, spontaneity, energy, entropy, enthalpy, free energy (Gibbs)

What constitutes as potential energy in Gibbs equation?

delta H (change in enthalpy)

What constitutes as kinetic energy in Gibbs equation?

Temp * entropy

Conditions for an exergonic/spontaneous rxn:

- delta G < 0
- Entropy (S) must be VERY positive (kinetic > potential)
- maybe increase T

Conditions for an endergonic/nonspontaneous rxn:

- requires nrg input
- delta G > 0

What if delta G = 0?

equilibrium achieved

In a reaction coordinate graph, delta G is:

the difference b/w reactants and products

In a reaction coordinate graph, energy of activation (EA) is:

the difference between reactants and transition state

Catalysts functions:

- stabilize transition state
- reduce activation energy (speed up rxn)
- Can't be used up in rxn

Catalysts change the rxn's ____________ (thermodynamics/kinetics)?

kinetics

Why don't catalysts change rxn's thermodynamics?

can't make the rxn spontaneous therefore delta G unchanged

Methods to activate/regulate enzymes

- Phosphorylation
- Allosteric regulation
- Negative/positive feedback

What is a necessary mechanism needed in positive feedback?

Need an external regulator to set things back to baseline (basically a bigger negative feedback loop to regulate products)

Km:

[S] required to reached HALF of Vmax

What does Km measure?

enzyme to substrate affinity
- low Km = high affinity
- high Km = low affinity

What does Km depend on?

- pH
- temperature
- ionic strengths
- substrate concentration/nature

Competitive inhibition:

inhibitor binds to FREE ENZYME and competes with substrate via enzyme active site

How to undo competitive inhibition?

increase [S] (substrate concentration)

How does competitive inhibition affect Vmax and Km?

- Vmax stays the same
- Km increases

Why does Vmax stay unchanged for competitive inhibition?

The inhibitor binds reversibly, therefore the substrate can compete with it and the enzyme concentration isn't the issue, but substrate concentration is.

Why does Km increase for competitive inhibition?

takes more substrate to reach 1/2Vmax

What does competitive inhibition look like in Michaelis-Menten plot (with regards to Vmax and Km)?

- Same Y-intercept (Vmax)
- Steeper slope (decreased Km, therefore increased 1/Km)

Noncompetitive inhibition:

inhibitors bind @ allosteric site of FREE ENZYME to turn it off. Enzyme's active site shape doesn't change so substrate could still bind to it but there'll be no products made

Which forms of inhibition is reversible?
I. Competitive inhibition
II. Noncompetitive inhibition
III. Uncompetitive inhibition

A. I only
B. II and III only
C. I and II only
D. I and III only

D.

How does noncompetitive inhibition affect Vmax and Km?

- Vmax decreases
- Km unchanged

Why does Km stay the same in noncompetitive inhibition?

substrate can still bind to enzyme active site, but no products are made

What does noncompetitive inhibition look like in Michaelis-Menten plot (with regards to Vmax and Km)?

- Y-intercept INCREASE (Vmax decreased therefore 1/V increase)
- X-intercept unchanged (Km)

Uncompetitive inhibition:

inhibitor binds at ES-complex ONLY at enzyme's allosteric site therefore no products made. Substrate cannot leave once it binds to enzyme.

How to undo uncompetitive inhibition?

Once uncompetitive inhibitor leaves the enzyme allosteric site, product is made IMMEDIATELY

How does uncompetitive inhibition affect Vmax and Km?

- Vmax decrease
- Km decrease

Why does Km decrease for uncompetitive inhibition?

The uncompetitive inhibitor STABILIZES ES-complex therefore substrates are more likely to bind to enzyme, which lowers Km.

What does uncompetitive inhibition look like in Michaelis-Menten plot (with regards to Vmax and Km)?

Line shifts to the LEFT of uninhibited enzyme, parallel slope

Mixed inhibition:

Mixed inhibitor binds to EITHER:
- allosteric site of free enzyme and DOES change active site shape -> Vmax decrease, Km increase, OR
- ES complex (Vmax decreased, Km increased - uncompetitive inhibition)