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Biochem > Lecture 8: Mechanisms > Flashcards

Lecture 8: Mechanisms Flashcards

1
Q

What type of enzyme is HIV protease?

A

aspartyl protease

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2
Q

Why is HIV protease needed?

A

has to cut a polyprotein in order for individual proteins to do work for HIV

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3
Q

Where does HIV protease bind?

A

at locations with Phe and Pro

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4
Q

How does HIV mechanism work?

A

Water is activated to attack onto the carbonyl carbon to form a tetrahedral intermediate which collapses

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5
Q

How is water activated in HIV mechanism?

A

through base catalysis with an aspartyl residue

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6
Q

What are the aspartyl residues used for in HIV protease?

A

one is used for base catalysis

the other is used to stabilize the negative oxygen in the tetrahedral intermediate

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7
Q

Drugs to treat HIV

A

transition state analogs

have bulky groups like Phe and Pro

resemble a N-group and Carbonyl

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8
Q

Transition state analogs

A

resemble the transition state more than the ground state of the substrate

act as an inhibitor

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9
Q

Penicillin function

A

blocks transpeptidase which synthesizes the peptidoglycan cell wall

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10
Q

B-lactamase function

A

breaks down pencilin

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11
Q

Structure of peptidoglycan

A

has peptide chain and sugar part

amino acids are in the D-conformation

transpeptidase can bind onto the peptide chain

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12
Q

Transpeptidase function

A

binds onto peptidoglycan and allows peptides to covalently attach to each other and build the cell-wall

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13
Q

Structure of penicillin

A

4 membered ring (B-lactam ring) fused to a 5 membered ring (thiazolidine ring)

Structure mimics the natural substrate of transpeptidase

Rings look like a dipeptide with alternating N-groups and carbonyl groups

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14
Q

Augmentin

A

antibiotic combination

combines the use of regular penicillin with clavulanic acid

able to knock out transpeptidases and B-lactamases

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15
Q

Clavulanic acid

A

looks like penicillin and interacts with B-lactamase

however, clavulanic acid rearranges to form a structure that cannot be detached from B-lactamase

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16
Q

What is penicillin to B-lactamase?

A

a substrate

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17
Q

What is penicillin to transpeptidase?

A

an inhibitor

18
Q

Difference between B-lactamase and transpeptidase mechanism?

A

When B-lactamase forms covalent bond with penicillin it can detach

Water is allowed into B-lactamase’s active site which disbonds penicillin

19
Q

What type of enzyme is chymotrypsin?

A

a serine protease that helps break down proteins in foods

20
Q

Where does chymotrypsin cut?

A

after aromatic amino acids

Trp, Phe, Tyr

21
Q

What do you except the active site of chymotrypsin to be?

A

large and hydrophobic in order to allow aromatic rings into the active site

22
Q

Characteristics of chymotrypsin

A

in inactive form is a zymogen, chymotrypsinogen

has disulfide bonds

possesses a catalytic triad

23
Q

pH profile of chymotrypsin

A

activity is maximized at pH=8

Km is lowest (greater affinity) below 8

Kcat is fastest above 8

24
Q

How do you determine Kcat/Km?

A

use a low substrate concentration

25
Q

What happens to chymotrypsin at high pHs?

A

the salt bridge between Ile16 and Asp194 is disrupted

amino groups are deprotonated and the salt bridge goes away

this causes the active site to close

26
Q

What happens to chymotrypsin at low pHs?

A

His 57 is protonated and cannot interact in catalysis

27
Q

Overall mechanism of chymotrypsin

A

1) Acylation: peptide bond is cleaved and an ester linkage is formed between the protein and enzyme
2) Deacylation: ester linkage is hydrolyzed, enzyme is regenerated

28
Q

Catalytic triad of chymotrypsin

A

Ser, His, and Asp

29
Q

What does serine do in chymotrypsin intially?

A

acts as a nucleophile after the hydrogen is removed from the -OH group

30
Q

What does histidine do in chymotrypsin intially?

A

acts as a general base to pull off serine’s hydrogen

31
Q

What does aspartate do in chymotrypsin initially?

A

stabilizes the formation of a positive charge on histidine

makes it more likely for histidine to pull off a hydrogen

raises the pKa of histidine as histidine is more likely to have a proton

32
Q

What are the serines on either side of the active site used for?

A

as one performs a nucleophilic attack, the serine directly on the other side stabilizes the tetrahedral intermediate that forms

33
Q

Oxyanion hole

A

made up of serine and glycine to stabilize the tetrahedral intermediate in chymotrypsin

34
Q

Acylation step

A

serine attacks the carbonyl and forms a tetrahedral intermediate that is stabilized by the oxyanion hole

the tetrahedral collapses and the first product breaks off

the first product’s N-group is stabilized by the H+ on histidine (general acid catalysis)

left with the serine attached to the other part of the substrate

35
Q

Deacylation step

A

water comes into the active site and histidine acts as a general base to activate water

water attacks the carbonyl and forms second tetrahedral intermediate (stabilized by oxyanion hole)

positively charged histidine donates a proton back onto serine (general acid)

second tetrahedral product collapses and releases final product

36
Q

List all the intermediates of the chymotrypsin mechanism

A

1) Tetrahedral number 1
2) Acyl-enzyme
3) Tetrahedral number 2

37
Q

Are the transition states the intermediates of chymotrypsin mechanism?

A

No! Transition states are just when energy is the highest

Do not neccessarily line up with intermediates

38
Q

How many steps does HIV-protease have?

A

one step

39
Q

What is substrate of transpitadase?

A

the D-Ala D-Ala section of the peptide chain in peptidoglycan

40
Q

How many steps does transpeptidase have?

A

2 steps

first have an acylation step

then, have a deacylation step but with another amino acid (rather than water in chymotrypsin mechanism)

41
Q

What type of inhibitor is penicillin?

A

suicide inhibitor

forms a permanent covalent bond with transpeptidase

42
Q

What is B-lactamase mechanism very similar to?

A

chymotrypsin mechanism

almost the same two steps

Biochem (20 decks)

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