Biochem Lec 12- Enzymes IV: Catalytic Mechanisms & Chymotrypsin

What is Chymotrypsin?

• A digestive enzyme that is produced in the pancreas and is secreted into the small intestine

• It aids in the digestion of food proteins by catalyzing the hydrolysis of peptide bonds

What are enzymes that breakdown proteins by hydrolyzing the peptide bonds called? (i.e. Chymotrypsin)

Proteases

What type of protease is Chymotrypsin and what does it do?

Endoprotease → cleaves peptide bonds inside the polypeptide chain

What is Chymotrypsin specific for?

It is highly specific for protein substrates based on the amino acid R-group on the amino terminal side of the cleaved peptide bond (scissile peptide bond)

What peptide bonds does Chymotrypsin hydrolyze?

Peptide bonds on the carboxyl terminal side of aromatic (trp, phe, tyr) or methionine (met) residues

Chymotrypsin’s active site contains a special residue. What is it? Describe it.

Ser 195 → different from the other serine residues in the enzyme and is a highly reactive nucleophile

Enzymes with reactive serines are susceptible to what? What are they and what do they do?

organofluorophosphate neurotoxins → irreversibly inactivate these enzymes by covalent modification of highly reactive serines

Ex. diisopropylfluorophosphate (DIPF) and sarin

These compounds irreversibly inactivate these enzymes by covalent modification of highly reactive serines

Why is serine 195 reactive?

Because of two other active site amino acids: His57 and Asp102

What AA’s make up the “catalytic triad” of chymotrypsin?

Ser195, His57, and Asp102

What AA is Ser195 H-bonded to? What effect does this have on Ser195?

Ser195 is H-bonded to His57→ this increases the deprotonation of the Ser hydroxyl group, making it more strongly nucleophilic.

What property of His57 allows it to more readily promote the removal of the Ser195 -OH proton?

It is more basic than normal histidines→ pKa is >7.5

What AA does Asp102 stabilize? How does it stabilize this AA? What effect does this have on the AA?

Asp102 stabilizes His57 because the neg. charge on Asp102 forms ionic interactions with the pos. charge on His57. This increases His57’s properties as a base→ stabilizes the pos. charge on His57 when it accepts a proton from Ser195→ increases basicity by stabilizing His57, making it a stronger proton acceptor

In what class of enzymes is the catalytic triad?

Serine proteases and hydrolases

Serine proteases and hydrolases like Chymotrypsin have three conserved features in their active sites. What are they?

1. The catalytic triad

2. The S₁ pocket

3. The oxyanion hole

What is the role of the catalytic triad during catalysis?

Catalyzes the hydrolysis of the peptide bond in two steps

What is the role of the S₁ pocket during catalysis?

Binds and positions the peptide substrate so that the catalytic triad can work

What is the role of the oxyanion hole during catalysis?

Stabilizes the transition state via hydrogen bonding during the reaction

The chymotrypsin reaction is an example of what type of catalysis?

Covalent catalysis

The chymotrypsin reaction proceeds through ___ steps. What are these steps?

2

1. Formation of acyl enzyme intermediate

2. Hydrolysis of acyl enzyme intermediate

What occurs during the first step of the chymotrypsin reaction?

Reactive serine cleaves peptide bond and releases the amino component. The carboxyl component remains attached to enzyme as an “acyl enzyme” intermediate

1. Substrate binding

• Peptide substrate sits in active site

• Its P₁ aromatic side chain fits into S₁ pocket→ aligns peptide bond being cleaved next to Ser195

2. Activation of Ser195/Nucleophilic attack

• His57 acts as base→ accepts proton from -OH of Ser195→ made possible by Asp102 stabilizing His57→ makes it more basic

• Deprotonated Ser195 (strong alkoxide ion) attacks carbonyl of peptide bond

• Forms tetrahedral intermediate at carbonyl carbon (stab. by oxyanion hole)

3. Collapse of tetrahedral intermediate

• Oxyanion reforms carbonyl double bond, forcing peptide bond to break

4. Release of amine component

• His57 donates a proton to leaving group (the amine end of the peptide)

• Amine product diffuses away, leaving the acyl-enzyme intermediate

What happens during the second step of the chymotrypsin reaction?

5. Water binding

• A water molecule enters active site and takes position once held by amine product

• His57 orients water molecule for next step

6. Nucleophilic attack of water on the acyl-enzyme intermediate

• His57 accepts proton from water→ forms hydroxide anion

• Hydroxide anion attacks carbonyl carbon of acyl-enzyme intermediate

• Forms second tetrahedral intermediate (stab. by oxyanion hole)

7. Collapse of second tetrahedral intermediate

• Second tetrahedral intermediate collapses, reforming carbonyl double bond

• Bond between Ser195 and substrate breaks

• His57 donates proton back to Ser195, reforming its -OH

8. Release of carboxylic acid component

• The carboxylic acid product (the C-terminal fragment of the substrate) is released

• Enzyme is fully regenerated/ready for another catalytic cycle

Full chymotrypsin mechanism

What AA is chymotrypsin highly specific for?

The AA residue found on the amino terminal side of the peptide bond (P₁ residue)

Substrate specificity is accomplished by what?

The S₁ pocket→ S₁ pocket binds the P₁ and positions substrate

What is the chymotrypsin S₁ pocket lined with? How does this accommodate substrate binding?

Hydrophobic residues→ accommodates substrate binding by interacting with the aromatic R-groups of the P₁ amino acid residue.

Different proteases and hydrolases contain a common catalytic feature of the catalytic triad, reactive Ser, and oxyanion hole. What is this?

Specificity for different substrates achieved by different chemical complementarity of the S₁ pocket.

What is the location/function/P₁ specificity of chymotrypsin?

Location→ small intestine

Function→ protein/substrate digestion

P₁ specificity→ Aromatic AA’s and Methionine

What is the location/function/P₁ specificity of trypsin?

Location→ small intestine

Function→ protein substrate digestion

P₁ specificity→ positively charged AA’s (Arg and Lys)

What is the location/function/P₁ specificity of elastase?

Location→ pancreas and neutrophils (type of white blood cell)

Function→ breakdown of connective tissue

P₁ specificity→ small AA’s (Ala, Ser, Gly)

Site-directed mutagenesis and enzyme kinetics reveal what?

The critical nature of each residue of the active site catalytic triad. It shows that mutating any of the three residues drastically reduces catalytic efficiency→ triad is necessary for efficient catalysis.