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MBB 324 > Lecture 9 > Flashcards

Lecture 9 Flashcards

1
Q

Draw the general mechanism of peptide bond hydrolysis

A

Slide 2

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2
Q

Where does the substrate sits in trypsin classic serine protease

A

The substrates sits in a groove in the enzyme surface

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3
Q

What are the 4 structural features involved in serine protease catalytic activity

A
  1. Catalytic triad
    - General acid/base chemistry
  2. Oxyanion hole
    - Binding of the transition state intermediate
  3. Nonspecific binding between mainchains
  4. Specificity pocket
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4
Q

Draw the 4 structural features involved in serine protease catalytic activity

A

Slide 4

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5
Q

What is the catalytic triad? Draw the catalytic triad. What AA’s does it involved?

A

Asp102, His57, Ser195

Slide 5

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6
Q

Draw the entire serine protease catalytic mechanism

A

7 drawings

  1. Enzyme-substrate complex
  2. Transition state 1
  3. Product 1 formation
  4. Acyl-enzyme intermediate
  5. Deacylation: nucleophilic or deacylating or catalytic water
  6. Transition state 2
  7. Free enzyme

OR

  1. Enzyme-substrate complex
  2. Tetrahedral intermediate 1
  3. Acylenzyme
  4. Tetrahedral intermediate 2
  5. Free enzyme + product
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7
Q

How to regulate enzyme activity (e.g. of serine protease rxn)

A
  1. Inhibitors (reversible)
  2. Inactivators (irreversible)
  3. Chemical modifications
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8
Q

What are the 3 types of inhibitors

A
  1. Competitive (binds to E)
  2. Uncompetitive (binds to ES)
  3. Mixed (Noncompetitive, binds to both E and ES)
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9
Q

What are the 2 types of inactivators?

A
  1. Covalent modification

2. Suicide inhibitors

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10
Q

What are the 2 types of chemical modifications

A
  1. Phosphorylation

2. Proenzymes e.g. chymotrypsinogin

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11
Q

Draw the mechanism for competitive inhibitor

A

N/A

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12
Q

Draw mechanism of competitive inhibitor

A

Binds only to E

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13
Q

Where do competitive inhibitors bind?

A

I competes with S for the ACTIVE SITE or I can bind in ANOTHER LOCATION on E that renders E unable to bind S

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14
Q

Example of competitive inhibitor?

A

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin at S1 pocket

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15
Q

Draw mechanism of uncompetitive inhibitors

A

N/A

Binds only to ES

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16
Q

Draw mechanism of noncompetitive inhibitors

A

N/A

Binds to E and ES

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17
Q

Example of inactivation of enzymes thru covalent modification

A

Antibiotics:

Penicillin + transpeptidase
—-The inhibitor covalently modifies the enzyme—>
Inactivated enzyme

4-membered ring is a strain system. Mimics structure of peptide bond. Carbonyl of penicillin attacked by hydroxyl of transpeptidase.

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18
Q
  1. What does DFP stand for?
  2. What does it do?
  3. What is DFP soluble in?
  4. How can it be used as an affinity label for autoradiography?
  5. What is the half life of DFP?
  6. MW?
  7. What does it inhibit?
A
  1. Di-isopropylfluorophosphate
  2. Irreversible inhibitor (inactivator) of serine proteinases. Phosphorylates active serine of the proteinase.
  3. DFP is soluble in propan-2-ol
  4. [13C] and [3H] labelled DFP can be used as affinity labels for autoradiography
  5. 1 hour at neutral pH
  6. MW: 184.2
  7. Extremely hazardous cause inhibits acetylcholinesterase
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19
Q
  1. What does TPCK stand for?
  2. What does it do?
  3. What is DFP soluble in?
  4. How can it be destroyed?
A
  1. Tosyl-Phenylalanyl-ChloromethylKetone (1-Chloro-3-tosylamido-4-phenyl-2-butanone)
  2. Irreversible inhibitor (inactivator) of chymotrypsin. Requires an active enzyme and forms a covalent bond w/His of the active site
  3. Soluble in ethanol but poor soluble in water
  4. Destroyed in less than 30 min at pH 9
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20
Q

What are the 4 structures in E.coli signal peptidase?

A
  1. 2 anti-parallel B-sheet domains
  2. Disulphide bond
  3. SH3-like barrel
  4. Extended B-ribbon
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21
Q

Slide 25

A

N/A

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22
Q

Slide 26

A

N/A

23
Q

What molecule binds 5R B-lactams?

A

Class D B-lactamase

OXA-10

24
Q

What molecule binds 5S B-lactams

A

Signal peptidase

25
Q

Signal peptidases attack from ____ face vs. Ser/His/Asp proteases attack from ____ face

A

Signal peptidases attack from Si face vs. Ser/His/Asp proteases attack from Re face

26
Q

Describe the 4 groups in signal peptidase

A
  1. Covalent bond btwn Ser90 Og and C7 of penem
  2. Carbonyl oxygen (O8) of penem points into the oxyanion hole
  3. Lys 145 is completely buried
  4. Methyl group (C16) of penem mimics P1 Ala side chain
27
Q

Describe the signal peptidase active site

A
  1. Ser 90 Og / Lys 145 Nz = 2.9 Å
  2. Lys 145 only titratable residue near Ser 90
  3. Lys 145 also H-bonded to Ser 278
28
Q

Describe logos diagram

A

Slide 31

29
Q

Slide 32

A

N/A

30
Q

Draw scissile bond

A

N/A

31
Q

Describe the AA’s involved in oxyanion hole of signal peptide-signal peptidase complex

A

Ser 90 N, Ser 88 Ogamma, Ser 88 X1 = + 60 degrees

32
Q

Describe the mutational evidence for transition state stabilization by Ser 88

A

To test idea that serine 88 is involved in the oxyanion hole (aka stabilization of transition state), mutate serine 88 to diff residues. Change serine to alanine, and activity went away. Serine to cysteine (similar but not as good as H bond donor cause of electronegativity diff in sulphur to oxygen) had a little bit of activity. If replace serine with threonine, which has hydroxyl, activity is almost brought back to wild type level

33
Q

Slide 37

A

N/A

34
Q

3 names for deacylating water

A
  1. catalytic
  2. nucleophilic
  3. hydrolytic
35
Q

Properties of deacylating water

A
  1. Positioned within H-bonding distance to a general base.

2. At a suitable distance and angle of approach with respect to the carbonyl of the ester (Bürgi angle ~107°).

36
Q

Slide 39

A

N/A

37
Q

Slide 40

A

N/A

38
Q

What is Fo - Fc

A

difference density in the SPase active site

39
Q

What kind of inhibitor is Ro09484?

A

Irreversible inhibitor

40
Q

Lipohexapeptide Arylomycin A2 has similar ring structure to what antibiotic

A

vancomycin

41
Q

Properties of lipohexapeptide arylomycin A2?

A
  1. Fatty Acid @ N-term.
  2. 2 D-amino acids
  3. Methylated mainchain
  4. 3 residue macrocycle ring
  5. [3,3]-biaryl bridge (Tyrosine-like cross-link)
  6. MeHPG = 4-hydroxyphenylglycine
42
Q

Arylomycin A2

  1. Product of _______ synthesis
  2. Extracted/purified from ______
  3. Structural (____) chemical analysis
  4. Inhibitor of ______ (ki ~____uM)
A

Arylomycin A2

  1. Product of NON-RIBOSOMAL PEPTIDE synthesis
  2. Extracted/purified from STREPTOMYCES
  3. Structural (NMR) chemical analysis
  4. Inhibitor of SPase (ki ~1uM)
43
Q

What kind of inhibitor is arylomycin A2?

A

Competitive inhibitor (reversible)

44
Q

Arylomycin A2 contacts
How is it bound? How is it buried? In what fashion does it bind? Which terminus points into active site? What AA residue and number is —–?

A
  1. Noncovalently bound
  2. Buries 481 Å2 of surface on SPase
  3. Binds in parallel b-sheet fashion
  4. C-term points into active site
  5. O45 ………Ser90/Lys145/Ser88
45
Q

Where is the serpin superfamily present?

A

Present in eukaryotes, plants and viruses,

  1. Egg - the non-inhibitory serpin ovalbumin
  2. Beer - the barley Z protease inhibitor
  3. Blood - the principle protease inhibitors in human plasma
46
Q

Example of 4 serpins (protease inhibitors) in human plasma

A
  1. Antithrombin, controls the proteolytic coagulation cascade by INHIBITING FACTOR Xa
  2. C1-inhibitor, controls complement activation
  3. Plasminogen activator inhibitors (PAI-1 and PAI-2) control fibrinolysis
  4. Alpha-1-antitrypsin (alpha-1-proteinase inhibitor) modulates connective tissue restructuring.
47
Q

Serpins make up ~__% of the proteins in human plasma

A

10%

48
Q

A number of diseases share a common mechanism arising from similar mutations of diff serpins.

  1. Blood =
  2. Liver =
  3. Brain =
A
  1. Blood = Thrombosis
  2. Liver = cirrhosis
  3. Brain = dementia
49
Q

Describe the general serpin structure and how it changes with interaction w/protease

A
  1. A complex but CONSERVED TERTIARY structure
    - > 3 b-sheets + 9 a-helices.
  2. The inhibitory reactive site forms an EXPOSED LOOP with the P1-P1’ RESIDUES acting as a BAIT for the target PROTEASE.
  3. The interaction with the protease leads to the formation of an IRREVERSIBLE SERPIN-ENZYME COMPLEX and a dramatic STRUCTURAL CHANGE!
  4. The central b-sheet (sheet A) undergo considerable refolding.
50
Q

Describe the stability of serpin (native alpha-1-antitrypsin inhibitor) before and after binding to protease

A

Before, metastable (melting temp of 55 d C)

After forming serpin-protease complex: hyperstable (melting temp 120 d C)

51
Q

Slide 53

A

N/A

52
Q

Disruption of active site prevents ______

A

deacylation

53
Q

Describe the disruption of the active site, preventing deacylation.

A

Stretching of the active-site loop resulting in:

  1. Loss of the oxyanion hole
  2. The replacement of the STABILIZING ‘ACTIVATION’ SALE-BRIDGE btwn the N-terminal amine and Asp194 of trypsin with Lys328 of a1-antitrypsin.
  3. The catalytic triad of native trypsin is grossly DISTORTED in the complex with a shift of Ser195 from His57 well beyond hydrogen-bonding proximity.
54
Q

Structure of a serpin–protease complex shows inhibition by _____

A

deformation

MBB 324 (17 decks)

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