Dr Kevin Ahern Protease Mechanisms Aspartic Acid Aspartic Acid Histidine Cysteine Electron rich Metal Other Protease Families Cysteine Aspartyl Metallo Papain Cathepsin D Carboxypeptidase A ID: 775055
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Slide1
Enzyme Catalytic Mechanisms II
Dr. Kevin Ahern
Slide2Protease Mechanisms
Aspartic Acid
Aspartic Acid
Histidine
Cysteine
Electron rich
Metal
Slide3Other Protease Families
Cysteine
Aspartyl
Metallo-
Papain
Cathepsin D
Carboxypeptidase A
Cathepsin K
Cathepsin E
Collagenases
Calpain
Pepsin
Caspase
Slide4Cysteine Proteases
Substrate binding
Sulfur anion
Nucleophilic
attack
Peptide bond
breakage
Release of
first peptide
Entry of
water
Nucleophilic
attack
Bond
breakage
Movement of proton
Slide5Aspartyl Protease
Substrate
Aspartate side chains
Slide6Aspartyl Protease Mechanism of Action
Activation of water
Nucleophilic attack
Tetrahedral intermediate
Breakage of peptide bond
Slide7Carbonic 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
Slide8⇌
Carbonic Anydrase
CO2 + H2O ⇌ H2CO3 ⇌ HCO3- + H+
Zn
+2
His
His
His
O
H
H
⇌
Zn
+2
His
His
His
O
H
-
+ H
+
⇌
CO
2
Zn
+2
His
His
His
O
H
-
C
O
O
=
=
Zn
+2
His
His
His
O
H
C
O
O
=
-
H
2
O
HCO
3
-
`
`
`
Slide9Restriction Enzymes
Bacterial defenseRestriction/modification
Slide10Restriction Enzymes
Hind III
Slide11Restriction Enzymes
Cutting Mechanism
Slide12Activation of water
Nucleophilic attack
Magnesium ions
Electronic reorganization
Breakage of bond
E = Glutamic acid
D = Aspartic acid
Slide13Restriction Enzymes
Action of DNA methylase
Me
Me
Me
Me
X
Slide14Metabolic Melody
The Restriction Enzyme Song(to the tune of “Chim Chim Cher-ee”)Copyright © Kevin Ahern
I'm obsessed with A-A-G-C-T-TBecause it’s the binding site of Hin-d-IIICutting up DNA most readilyThe 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 soThey must fight off phages or they will not growProtection by chopping is their strategyAnd one of the cutters we call Hin-d-III On binding to A-A-G-C-T-TThe site recognition site’s bent easilyPhosphodiester attacking meanwhileHas 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