The next phase of our examination of chorismate requires that we learn a bit about antibodies.

• Antibodies (also called immunoglobulins) are glycoproteins synthesized by by the lymphocyctes of vertebrates in response to detection of "foreign" substances

• The foreign substances, called antigens, may be proteins, polysaccharides, or nucleic acids

• Each lymphocyte (white blood cell) and all its decendants synthesize the same antibody; hence, over time, as the animal is exposed to a wide variety of antigens, a large number of antibodies develops

• When an antigen - either new or previously encountered - binds to an antibody on the surface of a lymphocyte, the cell is stimulated to make more copies of the antibody for release into the blood stream

• These "free" antibodies bind additional antigens, forming insoluble complexes that mark the antigens for destruction by proteases or lymphocytes

The most abundant antibodies in humans are the immunoglobulin type G, IgG antibodies. These all have the same general structure, shown in the cartoon below:

• Two identical "heavy" chains are composed of four domains (blue)

• Two identical "light" chains having two domains are assembled by disulfide bonds (red)

• Each domain consists of about 110 amino acids

• The antigen binding sites are formed by the N-terminal ends of the light and heavy chains
  • This is where the variation in structure occurs that allows antibodies to recognize and bind tightly different antigens

• The binding usually is so tight that no water molecules will fit into the binding site along with the antigen

Here is a picture of a typical IgG antibody:

Human IgG B12, anti-HIV (1hzh)

A space-filling model shows how intertwined the chains are. The antigen binding site are the loop regions at top and bottom right of the first picture.

So what does this have to do with chorismate mutase? Well, small molecules generally are not antigens.

• They can be made to elicit an antibody by attaching them to a carrier protein.

• The shape-selective binding of chorismate and the transition state inhibitor make them excellent candidates for generation of antibodies
  • The antibody may use the same residues to bind the TS inhibitor as chorismate mustase

  • The possibility then exists that the antibodies may be able to catalyze the rearrangement

• Both antibodies successfully bind both chorismate and the inhibitor, and catalyze the rearrangement of chorismate!

• Both are enantioselective

• One of them accomplishes a rate enhancement of about 10⁵, only about 10² less than chorismate mutase itself.

The crystal structure of the FAB fragment of the less effective catalytic antibody (rate enhancement about 200), known as 1F7, has been determined [Science, 1994, 263, 646]:

Antibody 1F7 with Substrate Bound (1fig)	Active Site of 1F7

Most of the binding is with the heavy chain. Note the similarity of the active site residues to those in the enzymes!

• As in chorismate mutase itself, an Arg makes a charge-charge interaction with the bridge carboxylate

• However, no positive group is hydrogen bonded to the ether oxygen, accounting in part for the lower reactivity.

• Overall, the antibodies seem to have less substrate complementarity: that is, fewer charged and hydrogen-bonding groups in a position to influence the substrate conformation

Curiously, despite the smaller number of binding groups, as compared to the enzyme, the antibodies actually bind substrate more tightly, by a factor of about 5.

In general, the direct elicitation of antibodies from an immune system is not an efficient route to catalytic species.

• Immune systems are polyclonal: that is, they fashiion a large number of antibodies, that may vary significantly in structure and binding affinity, and hence in catalytic activity.

• However, in the past ten years, techniques for creating monoclonal antibodies have led to the development of a large number of antibody-based catalysts.