Enzymes, Chirality, and Inhibition

Chirality

Chirality is prevalent in nature due to life's evolution based on carbon, which forms four bonds leading to chiral centers.
Chiral centers result in molecules with non-superimposable mirror images (enantiomers).
This asymmetry affects reactivity and bonding, even when asymmetry is not complete.
Proteins are composed of L-amino acids, leading to specificity for one enantiomer.
The use of L-amino acids dictates the structure of proteins.
The L and D amino acids refract polarized light to different sides. L to the left and D to the right.
Enzymes composed of D amino acids would have opposite reactions.
Using biomolecules ensures safer products with only one enantiomer.

Example of stereospecificity: Aconitase enzyme reaction.
Only one of the two identical substituents (CH2COO-) reacts due to the enzyme's recognition of a triangular base formed by three substituents.
Triangulation concept defines specific regions and constraints in space within proteins.
The enzyme has an iron-sulfur cluster coordinated by cysteines that coordinate a water molecule.
A base removes a proton, and the hydroxyl group gets swapped.

Proteases recognize L-amino acids; peptides synthesized with D-amino acids are not easily degraded.
D-amino acid peptides remain in the body longer, allowing for lower doses and higher efficacy.

Chymotrypsin: One peptide activated by proteolytic cleavage into three chains (A, B, C) with five disulfide bonds.
Key amino acids: Histidine 57, Aspartate 102, Serine 195, and Glycine 193 (stabilizes intermediary state).
Transition state stabilization by glycine, acid-base catalysis by histidine, and covalent bond formation with serine.
Catalytic triad: Serine, histidine, and aspartate.
Chymotrypsin has a hydrophobic pocket that binds phenylalanine, tyrosine, or tryptophan.
Specificity is determined by the substrate binding pocket's characteristics (charge, size, polarity).
Serine activation involves histidine taking a proton from serine with aspartate's assistance.
The activated serine forms an oxyanion stabilized by glycine, a short-lived intermediate.
The tetrahedral intermediate then releases part of the peptide, forming an acyl-enzyme intermediate.
A water molecule, activated by histidine, then attacks the carbonyl to release serine, freeing the enzyme.
Bonds formed with serine, threonine, or tyrosine hydroxyl groups are easily reactive and short-lived.

Most enzyme-catalyzed reactions involve acid-base catalysis, nucleophilic attack, and electrophilic interactions.
Essential components:
- Imidazole (histidine) as a nucleophile.
- Uncharged amino groups (lysine, arginine).
- Sulfhydryl or oxygen to abstract protons.
- Carbonyl for electrophilic attack.
- Phosphate for phosphorylation (kinases and phosphatases).

Reaction with p-nitrophenyl acetate generates p-nitrophenol (measured as yellow) and acetic acid.
Monitoring the formation of p-nitrophenol indicates enzyme activity.
The burst phase: Enzyme saturation followed by a slow steady state (limited by acetic acid release).
The linear phase: Dictated by the slowest (rate-limiting) step.
Irreversible steps determine the reaction rate.

Competitive Inhibitors: Mimic the substrate and compete for the active site; should not react with the enzyme.
- They have association (Ka) and dissociation (Kd) constants.
- Slower "off rate" (dissociation) is desirable for effectiveness.
Allosteric Inhibitors: Bind elsewhere, causing conformational changes that disrupt the active site or inhibit substrate binding/product release.
Suicidal Inhibitors: React with and chemically modify the enzyme, forming irreversible covalent bonds.

PMSF inactivates serine protease by binding covalently to the catalytic serine residue, making it an irreversible inhibitor.
Aspirin: Irreversibly binds to an enzyme, forming a covalent bond and preventing substrate binding; the enzyme must be degraded and resynthesized. It slows down the effect of the enzyme in the body.

Enzymes are unique due to their specificity toward a single reactant, stemming from the chiral or prochiral center.
This allows reactions to be performed in only one way, generating one enantiomer.
Enzymes can be evolved to bind nearly any substrate and perform various reaction types with appropriate cofactors.