Lecture 11

Question 1

What are abzymes?

Answer 1

• Catalytic antibodies

- monoclonal Abs w/catalytic activity

Question 2

4 reasons to study catalytic Abs?

Answer 2

1. Insights into enzyme catalysis
2. Insights into the evolution of enzymes
3. Create designer enzymes
   - An alternate approach to “protein engineering”
   “…binding specificity and catalytic activity may be inextricably linked in enzymes,…” Linus Pauling
4. Insights into autoimmune diseases

Question 3

Describe the steps in producing monoclonal Abs

Answer 3

A means of obtaining large amounts of pure antibody (exactly the same amino acid sequence)

1. Inject antigen (TS-analog) into mouse.
2. Take B cells from spleen (which will make Ab but do not grow well in culture) and myeloma cells (cancerous mouse B cells) (which will grow well in culture but do not make Ab) and add polyethylene glycol (PEG) (which is used to fuse B cells and myeloma cells)
3. Hybrid cells (hybridoma cells) grow in culture and make Ab. Only fused hybridoma cells will survive
4. Cells put in media (called HAT) - unfused myeloma cells die (they lack an enzyme only available from B cells)
5. Separate hybridoma cells - 1 cell per well (Fas separation). Screen for the presence of Ab w/the desired properties
6. Purify identical Mab.

Question 4

Draw a plot showing the energy involved in catalyzing a chemical rxn

Answer 4

Slide 5

Question 5

Describe the 4 steps to find (select for) catalytic antibodies with hydrolase activity

Answer 5

1. Use a transition state analog as an antigen
2. Make monoclonal antibodies to the antigen
3. Look for activity in antibodies (selection with an assay)
4. Purify Fab fragment from antibody

Question 6

Slide 7

Answer 6

N/A

Question 7

Slide 8

Answer 7

N/A

Question 8

Slide 9

Answer 8

N/A

Question 9

Slide 10

Answer 9

N/A

Question 10

Slide 11

Answer 10

N/A

Question 11

The amino acid ester hydrolysis reaction catalyzed by antibody ___

Answer 11

17E8

Question 12

For the 17E8 Fab fragment with bound Ts-analog, the binding site mostly formed by ____

Answer 12

CDR3 (Complementarity-Determining Region)

Question 13

Slide 14

Answer 13

N/A

Question 14

Slide 15

Answer 14

N/A

Question 15

Slide 16

Answer 15

N/A

Question 16

Slide 16

Answer 16

N/A

Question 17

Slide 17

Answer 17

N/A

Question 18

Slide 17

Answer 18

N/A

Question 19

Slide 17

Answer 19

N/A

Question 20

Slide 17

Answer 20

N/A

Question 21

Slide 18

Answer 21

N/A

Question 22

Slide 18

Answer 22

N/A

Question 23

Slide 19

Answer 23

N/A

Question 24

Slide 19

Answer 24

N/A

Question 25

3 Important differences between this catalytic Ab and serine protease

Answer 25

1. Ser-His Dyad instead of Ser-His-Asp Triad
   - No buried Asp to increase basic character of His and neutralize + charged on His during rxn.
2. Oxyanion hole
   - Conformationally mobile cationic Lys e-amino
   - Instead of a neutral conformationally restricted mainchain NH
3. Binding pocket(s)
   - Ab contains only the S1 binding pocket
   - Serine proteases usually have S1 + S3 pockets