Lecture 9

Question 1

Draw the general mechanism of peptide bond hydrolysis

Answer 1

Slide 2

Question 2

Where does the substrate sits in trypsin classic serine protease

Answer 2

The substrates sits in a groove in the enzyme surface

Question 3

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

Answer 3

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

Question 4

Draw the 4 structural features involved in serine protease catalytic activity

Answer 4

Slide 4

Question 5

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

Answer 5

Asp102, His57, Ser195

Slide 5

Question 6

Draw the entire serine protease catalytic mechanism

Answer 6

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

Question 7

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

Answer 7

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

Question 8

What are the 3 types of inhibitors

Answer 8

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

Question 9

What are the 2 types of inactivators?

Answer 9

1. Covalent modification

2. Suicide inhibitors

Question 10

What are the 2 types of chemical modifications

Answer 10

1. Phosphorylation

2. Proenzymes e.g. chymotrypsinogin

Question 11

Draw the mechanism for competitive inhibitor

Answer 11

N/A

Question 12

Draw mechanism of competitive inhibitor

Answer 12

Binds only to E

Question 13

Where do competitive inhibitors bind?

Answer 13

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

Question 14

Example of competitive inhibitor?

Answer 14

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

Question 15

Draw mechanism of uncompetitive inhibitors

Answer 15

N/A

Binds only to ES

Question 16

Draw mechanism of noncompetitive inhibitors

Answer 16

N/A

Binds to E and ES

Question 17

Example of inactivation of enzymes thru covalent modification

Answer 17

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.

Question 18

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?

Answer 18

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

Question 19

1. What does TPCK stand for?
2. What does it do?
3. What is DFP soluble in?
4. How can it be destroyed?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

Slide 25

Answer 21

N/A

Question 22

Slide 26

Answer 22

N/A

Question 23

What molecule binds 5R B-lactams?

Answer 23

Class D B-lactamase

OXA-10

Question 24

What molecule binds 5S B-lactams

Answer 24

Signal peptidase

Question 25

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

Answer 25

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

Question 26

Describe the 4 groups in signal peptidase

Answer 26

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

Question 27

Describe the signal peptidase active site

Answer 27

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

Question 28

Describe logos diagram

Answer 28

Slide 31

Question 29

Slide 32

Answer 29

N/A

Question 30

Draw scissile bond

Answer 30

N/A

Question 31

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

Answer 31

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

Question 32

Describe the mutational evidence for transition state stabilization by Ser 88

Answer 32

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

Question 33

Slide 37

Answer 33

N/A

Question 34

3 names for deacylating water

Answer 34

1. catalytic 2. nucleophilic 3. hydrolytic

Question 35

Properties of deacylating water

Answer 35

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°).

Question 36

Slide 39

Answer 36

N/A

Question 37

Slide 40

Answer 37

N/A

Question 38

What is Fo - Fc

Answer 38

difference density in the SPase active site

Question 39

What kind of inhibitor is Ro09484?

Answer 39

Irreversible inhibitor

Question 40

Lipohexapeptide Arylomycin A2 has similar ring structure to what antibiotic

Answer 40

vancomycin

Question 41

Properties of lipohexapeptide arylomycin A2?

Answer 41

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

Question 42

Arylomycin A2 1. Product of _______ synthesis 2. Extracted/purified from ______ 3. Structural (____) chemical analysis 4. Inhibitor of ______ (ki ~____uM)

Answer 42

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)

Question 43

What kind of inhibitor is arylomycin A2?

Answer 43

Competitive inhibitor (reversible)

Question 44

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 -----?

Answer 44

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

Question 45

Where is the serpin superfamily present?

Answer 45

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

Question 46

Example of 4 serpins (protease inhibitors) in human plasma

Answer 46

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.

Question 47

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

Answer 47

10%

Question 48

A number of diseases share a common mechanism arising from similar mutations of diff serpins. 1. Blood = 2. Liver = 3. Brain =

Answer 48

1. Blood = Thrombosis 2. Liver = cirrhosis 3. Brain = dementia

Question 49

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

Answer 49

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.

Question 50

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

Answer 50

Before, metastable (melting temp of 55 d C) | After forming serpin-protease complex: hyperstable (melting temp 120 d C)

Question 51

Slide 53

Answer 51

N/A

Question 52

Disruption of active site prevents ______

Answer 52

deacylation

Question 53

Describe the disruption of the active site, preventing deacylation.

Answer 53

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 Asp 194 of trypsin with Lys 328 of a1-antitrypsin. 3. The catalytic triad of native trypsin is grossly DISTORTED in the complex with a shift of Ser 195 from His 57 well beyond hydrogen-bonding proximity.

Question 54

Structure of a serpin–protease complex shows inhibition by _____

Answer 54

deformation