Serine Proteases

Serine proteases

enzymes that cleave peptide bonds using active site Serine

What is the catalytic triad? (chymotrypsin numbering)

Ser195, His57, Asp102

Which residue is the nucleophile in serine proteases?

Seri195

Which catalytic triad residue acts as a general acid and general base?

His57

Which catalytic triad residue properly orients and stabilizes His57 through hydrogen bonding?

Asp102

Why is Asp102 buried in a hydrophobic pocket?

To increase its ability to stabilize and orient His57

Which catalytic residue attacks the peptide bond?

Ser195

What activates Ser195?

His57 removes its proton, producing Ser-O⁻

During the first step of catalysis, His57 acts as what?

general base

During collapse of the tetrahedral intermediate, His57 acts as what?

general acid

What intermediate forms after Ser195 attacks the peptide bond?

tetrahedral intermediate

What stabilizes the tetrahedral intermediate?

oxyanion hole

What is the oxyanion hole?

A pocket that stabilizes the negatively charged oxygen of the tetrahedral intermediate using hydrogen bonds

Which backbone amides form the oxyanion hole in chymotrypsin?

Gly193 and Ser195

What intermediate forms after the first product leaves?

acyl-enzyme intermediate

What is covalently attached to the enzyme in the acyl-enzyme intermediate?

substrate

Why must water enter the active site?

To hydrolyze the acyl-enzyme intermediate

During deacylation, what does His57 deprotonate?

water

What nucleophile attacks during deacylation?

Hydroxide

What happens after the second tetrahedral intermediate collapses?

enzyme is regenerated and the second product is released

Which catalytic strategies are used by serine proteases?

• Covalent catalysis

• General acid-base catalysis

• Transition-state stabilization

What determines substrate specificity in serine proteases?

binding pocket

Why does chymotrypsin prefer aromatic residues?

has a deep hydrophobic binding pocket

Why does trypsin recognize Lys and Arg?

Asp189 provides a negatively charged binding pocket that attracts positively charged side chains

What residue is in trypsin’s binding pocket?

Asp189

Which residues does elastase cleave after?

Small neutral residues:

• Gly

• Ala

• Val

• Ser

Why does elastase only bind small residues?

Its binding pocket is partially blocked by Val and Thr residues

What does DIPF do?

Irreversibly inhibits serine proteases

Which residue does DIPF modify?

Ser195

Why is DIPF useful experimentally?

It identifies the essential catalytic serine

What is TPCK?

affinity label that specifically modifies His57

Which catalytic residue was identified using TPCK?

His57

What is bovine pancreatic trypsin inhibitor (BPTI)?

protein inhibitor that prevents premature trypsin activity in the pancreas

Why is BPTI such a strong inhibitor?

It mimics the tetrahedral intermediate and binds extremely tightly, preventing water from accessing the active site

What is the approximate dissociation constant (Kd) of BPTI?

10^-13 M

What is a zymogen?

inactive enzyme precursor activated by proteolytic cleavage

Which enzyme activates trypsinogen?

Enteropeptidase

How is trypsinogen activated?

Cleavage after Lys15 by enteropeptidase

Once formed, what can trypsin activate?

Additional trypsinogen molecules

What is autocatalysis?

enzyme activates more molecules of its own zymogen

Which enzyme first activates chymotrypsinogen?

Trypsin

What is the active form of chymotrypsin?

α-Chymotrypsin

What is the zymogen form of elastase?

Proelastase

Which enzyme activates proelastase?

Trypsin

Which blood-clotting enzyme is a serine protease?

Thrombin

What does thrombin convert?

FIbrinogen → Fibrin

Why does fibrin aggregate after thrombin cleavage?

Thrombin removes a negatively charged peptide, allowing fibrin molecules to polymerize into fibers

What residue performs nucleophilic attack during acylation?

Ser195

What residue performs nucleophilic attack during deacylation?

OH⁻ generated from water

Which residue is the general base in both halves of the mechanism?

His57

Which residue stabilizes His57?

Asp102

Which structure lowers activation energy by stabilizing the transition state?

Oxyanion Hole

How do enzymes bind tighter to the transition state?

thru the Oxyanion Hole

What does DIFP do to all Serine Proteases?

irreversibly inhibit

How was Ser195 found to be essential?

chymotrypsin treated with DIFP

How does DIFP inhibit Ser195?

Ser–O⁻ attacks the electrophilic phosphorus atom of DIPF

What is an affinity label?

a molecule that looks like the enzyme's normal substrate but contains a highly reactive chemical group

What makes an affinity label different from the enzyme’s substrate?

highly reactive chemical group

What is the affinity label used for chymotrypsin?

TPCK

How does the structure of TPCK help it bind to active site?

resembles phenylalanine, an amino acid that chymotrypsin normally recognizes

Which amino acid does TPCK inhibit?

His57

How was His57 identified as an essential catalytic residue?

TPCK covalently alkylates His57, irreversibly inactivating the enzyme

What happened to the enzyme when Asp102 was replaced?

not inactive but lost catalytic power

Where is the catalytic triad located?

within the active site, adjacent to specificity pocket

What are the 3 components of the active site?

catalytic triad, specificity pocket, oxyanion hole

What type of enzymes are Serine proteases?

hydrolases (E.C.3.)

When can Trypsin, Chymotrypsin, and Elastase not cleave?

if scissile bond is next to Proline

What is an artificial substrate?

a molecule that isn't the enzyme's natural substrate but is designed to be hydrolyzed by the enzyme

What is p-Nitrophenylacetate?

artificial substrate of Serine Proteases

What does Chymotrypsin hydrolyzes p-nitrophenylacetate into?

p-Nitrophenol

Why was p-nitrophenylacetate used?

its product, p-nitrophenol, strongly absorbs at 400 nm, allowing the reaction to be monitored spectrophotometrically