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Enzyme Mechanism

The document discusses enzyme mechanisms, focusing on serine proteases, their catalytic mechanisms, and specificity in peptide bond cleavage. It outlines the steps involved in the catalytic process, including substrate binding, formation of intermediates, and release of products. Additionally, it covers various protease families, their inhibition, and the role of restriction enzymes in bacterial defense.

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2 views

Enzyme Mechanism

The document discusses enzyme mechanisms, focusing on serine proteases, their catalytic mechanisms, and specificity in peptide bond cleavage. It outlines the steps involved in the catalytic process, including substrate binding, formation of intermediates, and release of products. Additionally, it covers various protease families, their inhibition, and the role of restriction enzymes in bacterial defense.

Uploaded by

ლევან გულუა
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Enzyme Mechanisms

Biochemistry Free For All


Serine Proteases

• Cleave Peptide Bonds


• Specificity of Cutting
• Common Active Site
Composition/Structure
• Mechanistically Well Studied

Chymotrypsin
Chymotrypsin Catalysis

H2O
Chymotrypsin

Yellow Color

N-Acetyl-L-phenylalanine p-nitrophenyl ester p-nitrophenolate


Serine Proteases
Serine Proteases Folded Polypeptide Chain of Enzyme
• Catalytic Mechanism

Substrate for Enzyme


Catalytic Triad of Active Site
Serine Proteases
• Catalytic Mechanism

Region of Enzyme That


Determines What Substrate
the Enzyme Binds
Serine Proteases
• Catalytic Mechanism
2. Structural Changes Induced by Binding
Change Electronic Environment of Catalytic Triad

1. Binding of Substrate Stimulates


Slight Structural Changes
Serine Proteases
• Catalytic Mechanism

N abstracts Proton From


Serine’s Side Chain,
Creating Alkoxide Ion
Serine Proteases
• Catalytic Mechanism

Alkoxide Ion Makes Nucleophilic Attack


on Carbonyl Carbon of Peptide Bond
Serine Proteases
• Catalytic Mechanism Peptide Bond Broken as
N Binds to H on Histidine

Tetrahedral Intermediate
Stabilized by Oxyanion Hole
Serine Proteases
• Catalytic Mechanism Half of Polypeptide Released from Enzyme

Other Half of Polypeptide


Covalently Linked to Serine

Fast Phase of Catalysis Completed


Serine Proteases
• Catalytic Mechanism
2. Nitrogen Attacks Proton of Water

3. Hydroxide Attacks
Carbonyl Carbon

1. Water Enters Active Site


Serine Proteases Oxygen Abstracts H on N-Group,
• Catalytic Mechanism
Breaking Bond with Serine

Oxyanion Hole Stabilizes


Tetrahedral Intermediate
Serine Proteases
• Catalytic Mechanism

Slow Phase of Catalysis Complete


Serine Proteases
• Catalytic Mechanism Enzyme Returned to Original State

Other
Polypeptide
Fragment
Released

Cycle Complete. Enzyme Ready to Start Anew


Serine Proteases
• Catalytic Mechanism

1. Binding of substrate to S1 pocket


2. Formation of alkoxide ion
3. Nucleophilic attack
Stabilization of intermediate
4. Breakage of peptide bond
5. Release of peptide 1
6. Entry and activation of water
7. Release of peptide 2 from enzyme
S1 Pockets and Specificities

Lysine Phenylalanine Glycine


Arginine Tryptophan Alanine
Tyrosine Valine
Serine Proteases
• Site-directed mutagenesis
• No mutation - activity = 100
• Serine to alanine - activity = 0.00001
• Histidine to alanine - activity = 0.00001
• Aspartic acid to alanine - activity = 0.001
• All three to alanine - activity = 0.00001
• No enzyme - activity = 0.000000001
Protein Cleavage Agents

Subtilisin - C-terminal side of large uncharged side chains


Chymotrypsin - C terminal side of aromatics (Phe, Tyr, Trp)
Trypsin - C-terminal side of lysine and arginines (not next to proline)
Carboxypeptidase - N-terminal side of C-terminal amino acid
Elastase - Hydrolyzes C-side of small AAs (Gly, Ala)
Cyanogen Bromide (chemical) - Hydrolyzes C-side of Met
Inhibiting Protease Action

Serpins = Serine Protease Inhibitors

α-1-antitrypsin
Reactive oxygen species
Prevalence of α-1-antitrypsin deficiency

α-1-antitrypsin deficiency by %
Protease Mechanisms
Aspartic Acid
Histidine
Electron rich

Metal
Aspartic Acid Cysteine
Other Protease Families

Cysteine Aspartyl Metallo-

Carboxypeptidase
Papain Cathepsin D
A

Cathepsin K Cathepsin E Collagenases

Calpain Pepsin

Caspase
Substrate binding

Sulfur anion
Cysteine Proteases
Nucleophilic
attack

Bond
Peptide bond
breakage
breakage

Movement of proton

Nucleophilic
Release of
attack
Entry of first peptide
water
Aspartyl Protease

Substrate Aspartate side chains


Nucleophilic attack
Activation of water
Aspartyl Protease Mechanism of Action

Tetrahedral intermediate

Breakage of peptide bond


Carbonic Anydrase
CO2 + H2O ⇌ H2CO3 ⇌ HCO3- + H+

pH Activity
5.9 10,000
6.3 140,000
7.0 410,000
7.5 700,000
7.9 810,000
8.1 900,000
9.0 1,000,000
Carbonic Anydrase

CO2 + H2O ⇌ H2CO3 ⇌ HCO3- + H+


HCO3-
` H2O

` `
+ H+ O
H H O
H H

=
H

=
O O
- CO2 - C
O O C
⇌ ⇌ ⇌ O-

=
Zn +2
Zn +2
Zn +2
O Zn+2
His His His His His His His His His His His His
Restriction Enzymes

Bacterial defense
Restriction/modification
Restriction Enzymes
Hind III
Restriction Enzymes

Cutting Mechanism
Activation of water E = Glutamic acid
D = Aspartic acid
Nucleophilic attack

Magnesium ions Breakage of bond

Electronic reorganization
Restriction Enzymes

Action of DNA methylase


Me Me

Me Me

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