Catalytic Antibody – AZ-28 Oxy Cope Rearrangement Biocatalyst Presented By: Woo-Jin Yoo CHEM*4450...
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Transcript of Catalytic Antibody – AZ-28 Oxy Cope Rearrangement Biocatalyst Presented By: Woo-Jin Yoo CHEM*4450...
Catalytic Antibody – AZ-28 Oxy Cope Rearrangement Biocatalyst
Presented By: Woo-Jin Yoo
CHEM*4450 – Biochemistry and Structure of Macromolecules
Seminar Series
Seminar Outline
1. Introduction2. Implications For Transition-State Analogs And Catalytic Antibodies3. AZ-28: Oxy-Cope Rearrangement Catalytic Antibody4. Structure And Function Relationship Of AZ-28 With Transition State Analog5. Structure And Function Relationship Of AZ-28 And Germline Antibody6. Concluding Remarks7. References
Introduction
Rationale for the development of biocatalyst
Advantages: • high regio- and stereoselectivity• environmentally friendly• non-toxic
Biocatalysts: enzymes, bioengineered microorganisms, ribozymes, catalytic antibodies
Advantage of catalytic antibodies:
Potential to design a biocatalyst for a specific reaction
Implications For Transition-State Analogs And Catalytic Antibodies
How do enzymes accelerate chemical reactions?
For one substrate to product situation:
Corresponding Energy Diagram:
Enzymes accelerate chemical reactions by stabilizing the transitionstate.
What are the available physical/chemical forces available forbinding and catalysis?
•Van der Waal
•Hydrogen bonding
•Hydrophobic effect
•Ionic Interactions
Designing a protein to catalyze a chemical reaction:
Idea: Proteins which binds transition state strongly should be able to catalyze a chemical reaction
Application: Immunize an animal using a transition state analog as a hapten to form antibodies that bind to the transition state analog
1. Determination and synthesis of the hapten (transition state analog)
2. Attachment of hapten to a macromolecule
BSA = Bovine Serum AlbuminKLH = Keyhole Limpet Hemocyanin
AZ-28: Oxy-Cope Rearrangement Catalytic Antibody
Background Information
• rearrangement is based on a diradical, cyclohexane TS intermediate• driven by the formation of a keto-enol compound
Why is this reaction slow/not possible at room temperature?
• transition state is a six-membered ring in the chair conformation• in solution, there are possible rotation of sigma bonds
Hapten design: Rationale
• transition state is a cyclohexane intermediate• strong preference for aromatic rings for catalytic antibodies• CONH(CH2)3COOH – is the tether to BSA/KLH
Structure And Function Relationship Of AZ-28 With Transition State Analog
AZ-28: Unliganded Mature Oxy- Cope Catalytic Antibody
Binding interactions between AZ-28 and Transition State Analog
5-phenyl group
• at the bottom of cavity• surrounded by large number of aromatic and hydrophobic residues• -stacking with H103His
2-phenyl group
• at the opening of the binding pocket• orientation is fixed by -stacking with H96His and van der waal interaction with side chain of L91Tyr
Cyclohexane ring
• position fixed by H-bonding with OH group and the imidazole ring of H96His• van der waal contact with L33Asn H101Asp
Mechanistic proposal for the Oxy-Cope rearrangement with AZ-28
1. Entopic Effect
Extended conformation is fixed into the energetically unfavorable conformation by thebinding site of the antibody
G = H - TS
Fixing conformation = S , G
2. Electronic Effect
Side chain of H96His and H-bonding of bridging water to H50Glu increases theelectron density of oxygen
Increased electron density on the oxygen will increase the rate of Oxy-Coperearrangement
Structure And Function Relationship Of AZ-28 And Germline Antibody
Fab of Mature Antibody Fab of Germline Antibody
What the heck is going on?
AZ-28 binds the TS analog more tightly than the germline antibody, but thegermline antibody is a better catalyst
Reason for increased catalysis of germline antibody
Recall:
The transition state of the Oxy-Coperearrangement is a diradical
The radical can be stabilized by the aromatic group
Molecular orbital reasons for increased catalytic activity
Note: stabilization of the transition state decreases the energy requirements for catalysis
When radical is in the sameplane as aromatic ring
When radical is perpendicularto the aromatic ring
• The germline antibody fixes the TSA so that the aromatic rings are 63.2o (5-phenyl) and 57.9o (2-phenyl) to the cyclohexane framework• AZ-28 fixes the TSA so that the aromatic rings are 81o (5-phenyl) and 85o (2-phenyl) to the cyclohexane framework
Structural reasons for difference in aromatic ring angle between AZ-28 andgermline antibody
Primary sequence difference
Structural basis for catalytic properties of germline and affinity matured antibody
Only L34 amino acid residue is at the active site
AZ-28 – L34AsnGermline – L34Ser
Liganded AZ-28 – 2.6 ÅUnliganded AZ-28 – 3.2 Å
Liganded Germline – 3.0 ÅUnliganded Germline – 3.7 Å
Increased flexibility of activesite for germline antibodylowers the rotational barrierfor the C2-phenyl
Result:
Concluding Remarks
Comments: AZ-28 binds more tightly to the TSA than germline antibody. However, germline antibody is a better catalyst
Reason: Flaw in the design of TSA. True TS possess sp2 carbons attached to the aromatic groups. TSA have sp3 carbons and in solution, the aromatic groups prefer to be perpendicular to the cyclohexane framework
References
1. Ulrich, H.D., Mundorff, E.C., Santarsiero, B.D., Driggers, E.M., Stevens, R.C., and Schultz, P.G. (1997) Nature 389, 271-275. 2. Driggers, E.M., Cho, H.S., Liu, C.W., Katzka, C.P., Braisted, A.C., Ulrich, H.D., Wemmer, D.E. and Schultz, P.G. (1998) J. Am. Chem. Soc., 120, 1945-1958.3. Mundorff, E.C., Hanson, M.A., Varvak, A., Ulrich, H.D., Schultz, P.G., and Stevens, R.C. (2000) Biochemistry 39, 627-632. 4. Braisted, A.C. and Schultz, P.G. (1994) J. Am. Chem. Soc., 116, 2211-2212.5. Mader, M.M., and Bartlett, P.A. (1997) Chem. Rev., 97, 1281-1301.