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

Example: Oxy-Cope rearrangement catalytic antibody AZ-28

1. Determination and synthesis of the hapten (transition state analog)

2. Attachment of hapten to a macromolecule

BSA = Bovine Serum AlbuminKLH = Keyhole Limpet Hemocyanin

3. Preparation of monoclonal antibodies

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

Comparison of AZ-28 with germline antibody

AZ-28 bound with TSAgermline bound with TSA

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

Electron density diagram of the active site for AZ-28

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.