Enzymes and Chymotrypsin Mechanism

Enzymes

Enzymes are mostly a large structure (blue) with a small active site (yellow).
The blue structure positions groups in the active site perfectly, controls the enzyme's activity through inhibitor binding, and facilitates subunit interactions in enzymes with quaternary structure.
Active site participants include amino acid residues, cofactors (metal ions), and coenzymes (small organic molecules).
Cofactors are usually metal ions and cannot be substituted by other ions with the same charge due to differences in their orbitals.
Coenzymes are small organic molecules, many of which are vitamins (e.g., NAD, FMN, Lipoic acid, biotin).
Some enzymes require both a cofactor and a coenzyme.

Catalysis

Catalysis involves the breaking and making of bonds, often through nucleophilic attack or acid/base catalysis.
Nucleophiles: Atoms with electron density that attack partial positive charges.
- Examples:
  - Cysteine (C): SH group can be deprotonated to form S-, a strong nucleophile.
  - Serine (S): OH group can be deprotonated to form O- (alkoxide), a strong nucleophile.
  - Lysine (K): NH2 form can act as a nucleophile.
  - Histidine (H): Must be in its base form (no positive charge on the imidazole ring) to be a nucleophile.
  - Water: Can act as a nucleophile, especially if deprotonated to form OH-.
General Acid Catalysts: R groups that donate a proton to speed up a reaction.
- Examples: Aspartic acid to cysteine (the seven guys in that row of amino acids), but they must get the proton back later.
General Base Catalysts: R groups that grab a proton to speed up a reaction and must give the proton off later.
- Same seven as above, minus arginine (arginine always has a proton).
Cofactors can help pull electron density away from a carbon, making it a better target for nucleophiles (e.g., Zinc with a +2 charge).
Coenzymes like thiamine pyrophosphate (TPP) can act as nucleophiles.

pH Importance

Everything on this slide depends on pH.
Cells regulate pH using buffers, with different compartments having different pH levels.
pH affects ionic and hydrogen bonds, influencing protein structure.
Many compounds in a cell have charges (e.g., amino acids, ATP), making them pH-sensitive.
Scientists must control pH in experiments to maintain enzyme function.
Never put your protein or enzyme just in pure water, unless you’re doing an experiment where you’re trying to kill the enzyme.

Chymotrypsin Mechanism

Chymotrypsin cuts on the carboxyl side of large aromatic groups (W, Y, F).
Goal: Understand how chymotrypsin recognizes the cut site, makes the cut, and performs the reaction so quickly.
Chymotrypsin is an enzyme that attacks proteins or peptides; the mechanism involves two substrates (peptide and water) and forms two products (two peptides).
The overall reaction: Peptide + H2O --> Two smaller peptides (Hydrolysis)
The mechanism occurs in two stages, but in reality, it is a blur and happens in about a millisecond.

Stage 1: Substrate Binding and Acyl-Enzyme Formation

The peptide (S1) enters the active site, encountering a hydrophobic pocket.
The hydrophobic pocket orients the carbonyl carbon of the target peptide bond.
The pocket is formed by nonpolar residues like valine, leucine, and isoleucine.
Serine (Ser195) acts as a nucleophile, attacking the carbonyl carbon.
Histidine (His57) acts as a general base, pulling a proton off the serine's hydroxyl group to make it a better nucleophile (alkoxide).
A transition state (TS1) is formed, with the oxygen of serine momentarily attached to the carbonyl carbon.
The histidine is in its base form to act as a general base catalyst.
Aspartate (Asp102) raises the pKa of histidine, making it a better base, and orients the histidine through hydrogen bonding.
The three residues (Ser195, His57, Asp102) form a catalytic triad.
At the end of stage one, part of the substrate remains attached to serine, forming an acyl-enzyme intermediate.
The first product (P1) diffuses away.

Stage 2: Deacylation

A water molecule (S2) enters the active site and attacks the carbonyl carbon of the acyl-enzyme intermediate.
Histidine (His57) acts as a general base, grabbing a proton from water to make it a better nucleophile (hydroxide).
A second transition state (TS2) is formed.
The bond between serine and the substrate is broken, releasing the second product (P2).
The enzyme returns to its original state, ready for another catalytic cycle.
Water is being consumed.

Spectrophotometry

These intermediates can be isolated by using very cold temperatures.
Instead of using a purely aqueous buffer, mix in some organic solvent like ethanol, propanol, something that is miscible with water to lower the freezing point.
Use really fancy spectrophotometers that are capable of seeing things in milliseconds or less.

Enzyme Kinetics and Transition States

Enzymes speed up chemical reactions without magic; they obey the laws of chemistry and physics.
Plotting free energy vs. reaction coordinate helps explain enzyme kinetics.
Substrates (S) and products (P) have innate energy levels due to their structures.
Enzymes do not change the energy of S or P.
Reaction coordinate: progress of the reaction
V0=k[s]

Activation Energy

Without an enzyme (blue curve), a high activation energy is required to reach the transition state.
The transition state is the most ordered position on the reaction coordinate, resembling both S and P.
The velocity of the reaction depends on the number of S molecules and a rate constant (k).
The rate constant includes an exponential term related to the activation energy.
Increasing temperature increases reaction rate, but most cells do not live in warm environments.
The change in free energy to reach the transition state (delta G double dagger) is positive and depends on changes in enthalpy (delta H double dagger) and entropy (delta S double dagger).
\Delta G^{\ddagger} = \Delta H^{\ddagger} - T\Delta S^{\ddagger}
Energy to get to the transition state can be in the form of heat, or in making S more ordered.

Enzyme Catalysis

Enzymes (red curve) lower the activation energy by ordering the substrate in the active site and providing R groups, coenzymes, and cofactors to facilitate the reaction.
The transition state for the enzyme-substrate complex is different from the transition state for the substrate alone.
How do enzymes manage to lower this energy? The active site orients the substrate.