1. Enzyme Catalytic Mechanisms II
Dr. Kevin Ahern

2. Protease Mechanisms Aspartic Acid Histidine Electron rich Metal
Cysteine
Electron rich
Metal

3. Other Protease Families
Cysteine
Aspartyl
Metallo-
Papain
Cathepsin D
Carboxypeptidase A
Cathepsin K
Cathepsin E
Collagenases
Calpain
Pepsin
Caspase

4. Cysteine Proteases Substrate binding Sulfur anion Nucleophilic attack
Bond
breakage
Peptide bond
breakage
Movement of proton
Nucleophilic
attack
Release of
first peptide
Entry of
water

5. Aspartyl Protease
Substrate
Aspartate side chains

6. Aspartyl Protease Mechanism of Action
Nucleophilic attack
Activation of water
Aspartyl Protease Mechanism of Action
Tetrahedral intermediate
Breakage of peptide bond

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

8. CO2 + H2O ⇌ H2CO3 ⇌ HCO3- + H+ ⇌ ⇌ ⇌ Carbonic Anydrase - - - HCO3- H2O`
HCO3-
H2O
+ H+ C O = O H H H H H = - - O O
CO2 O O C ⇌ ⇌ ⇌ -
Zn+2
His O
Zn+2
His
Zn+2
His
Zn+2
His

9. Restriction/modification
Restriction Enzymes
Bacterial defense
Restriction/modification

10. Restriction Enzymes
Hind III

11. Restriction Enzymes
Cutting Mechanism

12. Electronic reorganization
E = Glutamic acid
D = Aspartic acid
Activation of water
Nucleophilic attack
Magnesium ions
Breakage of bond
Electronic reorganization

13. Action of DNA methylase
Restriction Enzymes
Action of DNA methylase Me X

14. Metabolic Melody The Restriction Enzyme Song
(to the tune of “Chim Chim Cher-ee”) Copyright © Kevin Ahern
Metabolic Melody
I'm obsessed with A-A-G-C-T-T
Because it’s the binding site of Hin-d-III
Cutting up DNA most readily
The ends are not blunt when they’re cut up you see
Five prime overhangs of A-G-C-T
Bacteria don’t have an immune system so
They must fight off phages or they will not grow
Protection by chopping is their strategy
And one of the cutters we call Hin-d-III
On binding to A-A-G-C-T-T
The site recognition site’s bent easily
Phosphodiester attacking meanwhile
Has water behaving as nucleophile
To stave off the phage for a little while
Why don’t these enzymes cut cell DNAs?
The answer’s provided by a methylase
Adding a methyl group on top of what
The sequence these enzymes would otherwise cut
So cells get protected in this simple way
From nuclease chewing of their DNA
The phage is not lucky in most every case
Unless methylases win the enzyme race
If that happens then, the cell gets erased