BIOL115 - Lecture 10

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What is the formula of a single substrate reaction:
S P
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What is the formula of multiple substrate reactions?
A + B P + Q
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Do all enzymes use the same strategy to perform multi-substrate reactions?
No
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What are the two types of multiple substrate reactions?
Sequential displacement reactions and Double displacement reactions
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What are Sequential displacement reactions?
Reactions form a ternary complex i.e both substrates bind at once. Catalysis occurs after both substrates bind. It is a random order reaction, or an ordered reaction.
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What is a double displacement reaction?
Substituted enzyme intermediate is formed. Catalysis occurs after first substrates bind (ping pong mechanism)
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Give an example of a Sequentail displacement reaction:
E +S1 ES1 ES1S2 -> E + P1 + P2
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Give an example of something that experiences ordered mechanism:
Lactate dehydrogenase
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What is the ping pong mechanisms characterised by?
By the formation of a substituted enzyme intermediate (E')
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Why is double displacement known as the ping pong mechanism?
Because substrates bounce in and out
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Give an example of something that experiences a double displacement reaction:
Aspartate aminotransferase
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When is the Michaelis Menton model not valid?
If two substrates vary in concentration
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What does the Lineweaver Burk plot look like for sequential displacement reactions?
Similar to mixed inhibition, but increasing S2 decreases slope. Apparent Vmax increases and Km decreases.
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What does the Lineweaver Burk plot look like for double displacement reactions?
Similar to uncompetitive inhibition, increasing S2 decreases intercepts, apparent Vmax and Km increases.
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What is Chymotrypsin?
It is a proteolytic enzyme used from protein digestion in animals.
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Where does Chymotrypsin cleave peptide bonds?
It cleaves peptide bonds selectively on C terminal side of large hydrophobic amino acids (Trp, Tyr, Phe, Met).
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What is the formula for the chymotrypsin reaction
A P + Q
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Which residues are involved in catalysis?
We can use irreversible inhibitors to identify catalytic residues. Irreversible inhibitors bind covalently to the enzyme, and tend to react with the most reactive residue. The most reactive reside tends to be in the active site.
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What are 3 examples of irreversible inhibitors?
Group specific reagents, affinity labels, suicide inhibitors.
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What do group specific reagents do?
They react with specific amino acid side chains e.g. -OH, -SH
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What do Affinity labels do?
They're substrate analogues that bind to active site, like S, but then bind irreversibly and block the active site.
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What are suicide inhibitors?
They are substrate analogues that are converted into an affinity label by the action of the enzyme e.g. 5-flurouracil
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In group specific reagents, which serine is modified?
Serine 195 only. It is the most reactive of 27 serine's in chymotrypsin.
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What are the three things in the chymotrypsin catalytic triad?
His57, Asp102, Ser195
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What do His57 and Asp102 do for Ser195?
They increase the reactivity
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How does His57 increase the reactivity of Ser195?
It polarises the OH group of Ser195. So it acts as a general base catalyst by accepting H+ ion.
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When His57 polarises the OH group of Ser195, what happens to it?
It becomes a highly reactive alkoxide ion - a powerful nucleophile.
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How does Asp102 assist in making Ser195 more reactive?
It positions His57 and counters the positive charge.
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What are the steps of peptide ydrolysis by chymotrypsin?
1.) Subtrate binding, proton abstraction, 2.) Unstable tetrahedral transition state 3.) Acyl enzyme intermediate, release of first peptide 4.) Abstraction of proton from water by His57 5.) Second tetrahedral transition state 6.) Regeneration of as
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What does the substrate binding occur through?
Ra hydrophobic interactions. Proton abstraction from Ser195 forms alkoxide ion.Alkoxide ion attacks peptide carbonyl group, forming tetrahedral transition state.
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What is the next stage?
Proton transferred from His57 to C terminal NH of the peptide, cleaving the peptide bond, releasing the peptide, and forming the acyl-enzyme intermediate.
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What happens after that?
His57 binds a water molecule and removes a proton. The hydroxyl ion attacks the alkyl enzyme carbonyl group, forming a second tetrahedral transition state.
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Next?
Proton is transferred from His57 to Ser195, cleavage of the peptide bond occurs and the second peptide is released. The enzyme has regenerated
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Why do many proteolytic enzymes use a catalytic triad?
To achieve cleavage of the stable peptide bond.
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How many times has the catalytic triad evolved?
3 times.
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Site directed mutagenesis can be used to do what?
To dissect the importance of active site residues.
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Give 3 serine proteases:
Chymotrypsin, trypsin and elastase
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Where does Chymotrypsin cleave?
It cleaves after aa with large hydrophobic side chains (Trp, Tyr, Phe & Met)
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Where does Trypsin cleave?
It cleaves after aa with long, positively charged side chains (Arg & Lys)
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Where does Elastase cleave?
It cleaves after aa with small side chains (Ala & Ser)
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Which is the most economically important enzyme?
Subtillisin
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What are the uses of Subtillisin?
As an enzyme in washing powder and other detergents. Can stand up well to presence of surfactants that might denature many other enzmes.
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What are possible problems that can affect enzymes?
Susceptibility to oxidation by bleaching agents, inactivated at high temperatures, some limitations on substrate recognition.
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What can help overcome each of these problems?
Mutagenesis
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What can allow us to deduce the catalytic reaction mechanisms?
The use of inhibitors and 3D structural information
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What plays a critical role in catalysis?
Specific amino acid side chains.
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Substrate specificity can be governed by interactions where?
Adjacent to the active site
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Card 2

Front

What is the formula of multiple substrate reactions?

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A + B P + Q

Card 3

Front

Do all enzymes use the same strategy to perform multi-substrate reactions?

Back

Preview of the front of card 3

Card 4

Front

What are the two types of multiple substrate reactions?

Back

Preview of the front of card 4

Card 5

Front

What are Sequential displacement reactions?

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