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BB450 > Enzyme Classifications and Enzyme Catalytic Mechanisms > Flashcards

Enzyme Classifications and Enzyme Catalytic Mechanisms Flashcards

1
Q

What are EC1 Oxidoreductases?

A

Oxidation/reduction reaction catalysis

They include NAD+ and NADH

Ex: Malate Dehydrogenase

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2
Q

What are EC2 Transferases?

A

Transfer a functional group

A methyl or a phosphate group

Ex. Hexokinase

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3
Q

What are EC3 Hydrolases?

A

Hydrolysis of bonds

Requires water to break bonds

Ex. Proteases

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4
Q

What are EC4 Lyases?

A

Non-hydrolytic, non-oxidative breaking of bonds

Ex. Isocitrate Lyase

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5
Q

What are EC5 Isomerases?

A

Catalyze isomerization changes within a single molecule.

Ex. Phosphoglucoisomerase

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6
Q

What are EC6 Ligases?

A

Join two molecules by making covalent bonds

Ex. Argininosuccinate Synthetase

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7
Q

What are of Proteases?

A

Proteases catalyze the hydrolysis of peptide bonds in polypeptides.

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8
Q

What do Serine Proteases have?

A

They have a serine residue that is essential for catalysis

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9
Q

Why is breaking peptide bonds important?

A

It is important for digestion in humans and is also seen in lab work.

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10
Q

Why do serine proteases have specificity when cutting peptide bonds?

A

They will recognize specific amino acids and cut adjacent to those amino acids.

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11
Q

What do all serine proteases have in common?

A

They have a common active site

They have a common composition and structure

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12
Q

Chymotrypsin is a serine proteases with a S1 pocket that binds to?

A

(Broad, non-polar) Phenylalanine, Tryptophan, Tyrosine

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13
Q

Trypsin is a serine proteases with a S1 pocket that binds to?

A

Lysine, Arginine

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14
Q

Elastase is a serine proteases with a S1 pocket that binds to?

A

Glycine, Alanine, Valine

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15
Q

What is a characteristic of Chymotrypsin?

A

Very flexible

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16
Q

How can Chymotrypsin be studied?

A

Chymotrypsin can be studied using an artificial substrate which, when cleaved by the enzyme, releases a yellow product.

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17
Q

What is the Mechanism for Serine Proteases?

A
  1. Binding of substrate to S1 pocket
  2. Formation of alkoxide ion
  3. Nucleophilic attack on the carbonyl carbon
  4. The intermediate is stabilize in the process
  5. Breaking of peptide 1 (Finish Fast phase)
  6. Entry of water into the active site
    Release of peptide 2 from enzyme
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18
Q

What do you get a bend in the line when looking at Serine Proteases (color produced vs time)

A

You have a Fast phase and a Slow phase.

19
Q

What is occurs in the fast phase of the Serine Protease process?

A

In the Fast phase, the peptide bond is broken, part of the polypeptide chain is released and free, part of the polypeptide chain becomes covalently bonded to the enzyme.

20
Q

What is occurs in the slow phase of the Serine Protease process?

A

In the Slow phase, you have the rest of the polypeptide chain being released from the chymotrypsin enzyme because it’s covalently bonded.

21
Q

What makes up the catalytic triad of an active site?

A

Serine, Histidine and Aspartic acid

Those amino acids are not close in proximity.

22
Q

What has to occur for the catalytic triad to come together?

A

The enzyme has to fold to bring these amino acids close to form the active site, where the reaction takes place.

23
Q

What are the steps that bring the catalytic triad together?

A

Steps
1. Binding of Substrate stimulates slight structural change. This causes many things to happen.

  1. Structural change induced by binding change electron environment of catalytic triad.
    N abstracts proton from serines side chain, creating alkoxide ion.
    Alkoxide ion makes a nucleophilic attack on carbonyl carbon of peptide bond.
    The result of the attack is the break of a peptide bond.
24
Q

What is the Oxyanion hole pocket?

A

Provides an environment for a very unstable intermediate to fall apart.

The intermediate is the tetrahedral formation

So the oxyanion hole stabilizes the unstable intermediate and allows it to fall apart.

25
Q

What is the S1 pocket of an enzyme responsible for?

A

S1 pocket is what determines the specificity of the enzyme

26
Q

How does site-directed mutagenesis affect enzyme activity?

A 
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- 0.0000000001
27
Q

What are reagents that cleave proteins?

A

Substilisin: C-terminal side of large uncharged side chains

Chymotrypson: C terminal side of aromatics (Phe, Tyr, Trp)

Trypson: 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

28
Q

What are inhibiting protease actions?

A

So these are inhibitors that stop the action of proteases that block access to active site by other polypeptides

29
Q

What do Serpins do?

A

Serpins= Serine Protease Inhibitors

Ex. Alpha-1-antitrypsin

It inhibits elastase in your lungs, that allow you to break down proteins in bacteria, if you have too much elastase you get emphazema so if you have too much elastase your body produces Alpha-1-antitrypsin

Polypeptide chains that block the active site.

30
Q

What are reactive oxygen species?

A

Smokers produce a lot of hydroxyl radical.

They are very reactive, that will attack the sulfur of a methionine (creates sulfoxide), and there is a critical sulfur in Alpha-1-antitrypsin that allows it to bind to elastase, when sulfur gets oxidize, it stops Alpha-1-antitrypsin from attaching to elastase and that is why smokers get emphysema, because the elastase attacks the lungs.

31
Q

What are Aspartyl Proteases?

A

They have 2 side chains of aspartic acid. No catalytic triad.

One of the carboxyl groups becomes ionized and one does not.

Why would one ionize and the other does not?

The electronic environment is not the same as an aqueous environment, so basically things behave differently inside of a protein than they do outside of a protein.
Aspartic acid grabs a proton off water.

32
Q

What are Cysteine Proteases?

A

Don’t have catalytic triad, only have 2 amino acids involved.

Have a Cysteine and a Histidine

Similar to serine protease. Why?

Binding of proper substrate, causes the histidine to move closer to the cysteine.

33
Q

What are Metalloproteases?

A

Has common themes as other proteases.
They have a side chain that is electron rich (amino acid), it helps in the activation of water.
This is seen in serine proteases.
They also have a Metal. The metal helps hold the water, so that it can be activated.

34
Q

What’s in the cysteine protease family?

A

Cathepsin D

Cathepsin E
• All involved in signaling.

Pepsin
• Digestive enzyme, in your stomach that is active at pH of 1-1.2.

35
Q

What’s in the metallo- protease family?

A

Carboxypeptidase A
• Digestive enzyme, very digestive, it starts at the carboxyl end and starts to chew up amino acids away, one at a time.
• Trypsin breaks a peptide into fragments, and those fragments are broken down into individual amino acids by Carboxypeptidase A.

Collagenases
Enzymes that break down collagen, in digestion. When you eat food, you need to break up the connective tissue of the food you are eating.

36
Q

What is the mechanism used for cysteine proteases?

A

Enzymes that use cysteine in their active site
Process:
Substrate binding by SH- of cysteine to Histidine.

Sulfur anion
• The proton is removed from SH- just like the OH is removed in serine.

Nucleophilic attack
• -S attacks the carbonyl carbon.

Peptide bond breakage
• We have a tetrahedral intermediate, and the peptide bond falls apart.

Movement of proton, release of first peptide
• Proton moves from the histidine hydrogen to the amine group of the released peptide
COMPLETION OF FAST CYCLE

Entry of water
• The second peptide is attached to the sulfur just like in the serine protease.
• Water is activated by the histidine, activation requires removal of proton.

Nucleophilic attack
• This causes the hydroxyl to attack the sulfur- carbon bond.
• This forms the tetrahedral intermediate
• The peptide bond breaks.

37
Q

What is the mechanism used for aspartyl proteases?

A

o Substrate is bound to the enzyme
o Aspartate side chains (projecting upwards)

There is a water that fits between the aspartate side chains.

Process:
Activation of water
• Common step!

Carboxyl pulls a proton off water and the hydroxyl created

Reactive hydroxyl does the nucleophilic attack
• You don’t have 2 steps, because the water attacks and not an amino acids

The attack creates a tetrahedral intermediate.
• The bond breaks into two peptides, and you’re DONE

What’s the difference here?
• There isn’t a covalent bond because water is doing the attacking, the water gets attached, but its not physically attached.
• The peptide gets broken into two and that is it, so you have ONE STEP
• This is a simpler mechanism.

38
Q

What is the mechanism used for carbonic anydrase?

A

This is an enzyme that works incredibly fast, it is responsible for dissolving carbon dioxide in your blood.

Carbon dioxide can get attached to hemoglobin, it displaces protons and it goes back to the lungs where it gets released. THIS IS A MINOR WAY TO RELEASE CO2

THE MAJOR WAY TO RELEASE CO2
o The major way that CO2 gets moved is by HCO3-
o CO2 + H2O H2CO3 HCO3- + H+

CO2 is a poisonous gas!
o pH matters for this enzyme, max cap at pH 9.
It has the highest activity rate at this pH

Process:
You have Zn holding on to water. Zn is held by 3 different histidines.

Loss of H+
• Water needs to ionized, so increasing the pH helps the mechanism.

O- attacks the CO2
• O- attacks the CO2 because it is electron poor.

Water enters and HCO3- released

39
Q

What do restriction enzymes do?

A 
Enzymes cut DNA at specific sequences. 
The enzyme stops at a specific sequence and it stops there and bends it. 
Mechanism:
-Looks for specific sequence
-Bends DNA at specific DNA
40
Q

Give an example of a restriction enzyme?

A

Hind III (Restriction Enzyme)
o Cuts out 5’-AAGC-3’
o Cuts out 3’- CGAA- 5’

This leaves a staggered end.
o Has a lot of Glu and Asp so its very negative
o Bending of DNA allows for reaction to occur. It only occurs at the proper sequence.

Similar mechanism, you have activation of water.

41
Q

Where are restriction enzyme found?

A

Only in bacteria

42
Q

What do bacteria use restriction enzymes for?

A

Bacterial defense

Viruses attack bacteria, the bacteria uses these restriction enzymes to cut the DNA that comes in.

43
Q

How do bacteria protect themselves from cutting its own DNA?

A

Action of DNA methylase

It’s a restriction enzyme that protects the cell

The Methylase recognizes AAGC and adds Methyl groups so now the cellular DNA is protected.

It’s a race between what enzyme gets there first

BB450 (16 decks)

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