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 Enzyme Catalytic Mechanisms II  Enzyme Catalytic Mechanisms II

Enzyme Catalytic Mechanisms II - PowerPoint Presentation

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Enzyme Catalytic Mechanisms II - PPT Presentation

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

000 enzymes restriction nucleophilic 000 enzymes restriction nucleophilic attack bond protease breakage acid water peptide cut enzyme cathepsin aspartyl

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Presentation Transcript

Slide1

Enzyme Catalytic Mechanisms II

Dr. Kevin Ahern

Slide2

Protease Mechanisms

Aspartic Acid

Aspartic Acid

Histidine

Cysteine

Electron rich

Metal

Slide3

Other Protease Families

Cysteine

Aspartyl

Metallo-

Papain

Cathepsin D

Carboxypeptidase A

Cathepsin K

Cathepsin E

Collagenases

Calpain

Pepsin

Caspase

Slide4

Cysteine Proteases

Substrate binding

Sulfur anion

Nucleophilic

attack

Peptide bond

breakage

Release of

first peptide

Entry of

water

Nucleophilic

attack

Bond

breakage

Movement of proton

Slide5

Aspartyl Protease

Substrate

Aspartate side chains

Slide6

Aspartyl Protease Mechanism of Action

Activation of water

Nucleophilic attack

Tetrahedral intermediate

Breakage of peptide bond

Slide7

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

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

-

`

`

`

Slide9

Restriction Enzymes

Bacterial defenseRestriction/modification

Slide10

Restriction Enzymes

Hind III

Slide11

Restriction Enzymes

Cutting Mechanism

Slide12

Activation of water

Nucleophilic attack

Magnesium ions

Electronic reorganization

Breakage of bond

E = Glutamic acid

D = Aspartic acid

Slide13

Restriction Enzymes

Action of DNA methylase

Me

Me

Me

Me

X

Slide14

Metabolic 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