Lecture 11 - Serine Proteases

Irreversible Inhibitors and Chymotrypsin

Allow us to understand its mechanism; can react with DFP to generate peptides from inactive enzyme and sequence (identify residues - SERINE)

Catalytic Triad of Serine Proteases

1. Serine - reactive covalent nucleophile

2. Histidine - acid/base catalyst

3. Aspartate - electrostatically enhance serine’s nucleophilicity

5 Key Players in Serine Proteases

1. Serine

2. Histidine

3. Aspartate

4. Oxyanion hole

5. Specificity pocket

Serine Function in Serine Protease

VERY reactive, acts as a nucleophile to bind bulky hydrophobic group

Histidine Function in Serine Protease

Acid-base catalyst (acts as BOTH base and acid)

Aspartate Function in Serine Proteases

Electrostatic interactions allow it to create environment that enhance Serine’s nucleophilicity

Oxyanion hole function

Stabilizes transition state, its amide hydrogen interacts with oxyanion

Specificity Pocket role in Chymotrypsin

Creates hydrophobic effect due to hydrophobic amino acids

Serine becomes bound to…

Acyl group on peptide

In order to cleave molecule…

The enzyme MUST BE deacylated

Serine Proteases display what system?

Charge-relay system (Ser - His - Asp)

Evolution of Serine Proteases

• Divergent - evolved from same ancestor, similar structures but limited homology “CHYMOTRYPSIN-LIKE”

  • Elastase and trypsin

• Convergent - evolved independently from ancestor, no structural similarity

  • Subtilisins

ALL share the same mechanism!!

What enzymes were evolved divergently?

Elastase and trypsin

What enzymes were evolved convergently?

Subtilisins

What interacts with the specificity pocket?

The R-group on the carbonyl side of the broken bond

Chymotrypsin Specificity

Bulky, hydrophobic groups (Phe, Try, Tyr, Leu) - aromatic

Has a lower K_m for these molecules

Trypsin Specificity

Positively-charged amino acids (Arg, Lys)

Elastase Specificty

Small, neutral residues (Ala, Gly, Ser)

What bond stabilizes the specificity pocket?

Disulfide bond (S-S), salt bridge in trypsin

Carboxypeptidase A

Metallo-exopeptidase that cleaves hydrophobic amino acids from the C-terminus (carboxyl end)

Is carboxypeptidase an endopeptidase or exopeptidase?

EXOpeptidase

Carboxypeptidase A Key Players

1. Arginine (cation) - coordinates the C-terminal carboxyl group

2. Zn²⁺ - prosthetic group that stabilizes the transition

3. Hydrophobic pocket - binds side chain

Arginine function of Carboxypeptidase A

Cation that coordinates the carboxyl group on a peptide

Zn²⁺ Function in Carboxypeptidase A

Stabilizes transition state as an electrostatic catalyst and PROSTHETIC GROUP

How to differentiate Carboxypeptidase A vs. Serine protease

1. Key players

2. CPA is NOT acylated (no deacylation required)

3. CPA does NOT have burst-steady state kinetics

4. Exo vs. endopeptidase

Triose-Phosphate Isomerase

Near-perfect acid-base catalyst arranged as a b-barrel surrounded by a-helices

What molecules does TPI interact with?

Simple sugars to shuttle 2 protons between carbons

Why is TPI nearly perfect?

• Its 3 key players: Glu, His, and Lys act as optimal acid-base catalysts because they are CLOSE together

  • Allows for HIGH k_cat/K_m

Key players in TPI

Glu, His, Lys (all acid/base catalysts)

What happens if you replaced Glu with Asp?

The enzymatic efficiency of TPI decreases greatly, because residues now have MORE space