Question

Catalytic antibodies are prepared against a transition state analog of a substrate.

Do catalytic antibodies work because they maximize kcat or minimize Km? Justify answer.

Answer 1

Catalytic Antibodies: They are also known as abzymes.

Transition-state analogs are also ideal for generating catalytic antibodies (abzymes).

Antibodies (immunoglobins) may be created to recognize transition states, and thus function as catalysts for the reaction.

The transition-state analog acts as an antigen (immunogen) to generate the antibody.

An example of this process is the production of an antibody that catalyzes the insertion of an iron ion into the porphyrin plane, which must be bent in order to allow the iron to enter.

Normally, this step is catalyzed by ferrochelatase, the final enzyme in the production of heme. N-methylprotoporphyrin was found to resemble the transition state because N-alkylation bends the porphyrin, much like the ferrochelatase enzyme.

Therefore, an antibody catalyst was produced by using an N-alkylporphyrin as the immunogenic. The produced antibody is able to distort the porphyrin in order to facilitate the entry of ferrous iron.

Using a similar technique, antibodies that catalyze ester and amide hydrolysis, transesterification, and photo induced cleavage, among other reactions, have been developed.

         Transition-state analog

Ping Pong Mechanism

For this mechanism, an enzyme can be in two states. One of the states is labeled E and the other state that is also known as the intermediate and that is chemically modified is labeled E*.

In this mechanism, the first substrate (substrate A) binds to enzyme turning it into E* by the transfer of a chemical group to the active site and then the substrate is released.

Once substrate A is released, substrate is able to bind to the modified Enzyme (E*) forming the unmodified Enzyme once again (regeneration).

When a Line-Weaver Burk plot is graphed, two sets of parallel will be formed opposite of the Ternary Complex Mechanism. Specific enzymes that follow this mechanism include oxidoreductases and serine proteases.

Some of the serine proteases include the digestive enzymes of chymotrypsin and trypsin. For the example of the chymotrypsin, an acyl-enzyme is formed after the breakdown of the tetrahedral intermediate, which is formed after the nucleophilic attack of Ser to the carbonyl forming the intermediate.

Once the intermediate breaks down, the acyl-enzyme is formed which acts as the modified Enzyme (E*).

The acyl-enzyme however breaks down later into the intermediate complex as the amine group of the acyl-enzyme (E*) leaves and hydrogen functions as a nucleophile to attack the carbonyl forming the tetrahedral intermediate once again.

Catalytic antibodies are antibodies that can enhance a couple of chemical and metabolic reactions in the body by binding a chemical group, resembling the transition state of a given reaction.

Catalytic antibodies are produced when an antibody is immunized with a hapten molecule. The hapten molecule is usually designed to resemble the transition state of metabolic reaction.

Ordinarily, antibody molecules simply bind; they do not catalyze reactions.

However, catalytic antibodies are produced when animals are immunized with hapten molecules that are specially designed to elicit antibodies that have binding pockets capable of catalyzing chemical reactions.

For example, in the simplest cases, binding forces within the antibody binding pocket are enlisted to stabilize transition states and intermediates, thereby lowering a reaction's energy barrier and increasing its rate.

This can occur when the antibodies have a binding site that is complementary to a transition state or intermediate structure in terms of both three-dimensional geometry and charge distribution.

This complementarity leads to catalysis by encouraging the substrate to adopt a transition-state-like geometry and charge distribution.

Not only is the energy barrier lowered for the desired reaction, but other geometries and charge distributions that would lead to unwanted products can be prevented, increasing reaction selectivity.

Catalytic antibodies bind very tightly to the transition-state analog haptens that were used to produce them during the immunization process.

The transition-state analog haptens only bind and do not react with catalytic antibodies. It is the substrates, for example, the analogous ester molecules, that react.

For this reason, transition-state analog haptens can interfere with the catalytic reaction by binding in the antibody binding pocket, thereby preventing any substrate molecules from binding and reacting. This inhibition by the transition-state analog hapten is always observed with catalytic antibodies, and is used as a first level of proof that catalytic antibodies are responsible for any observed catalytic reaction.

The important feature of catalysis by antibodies is that, unlike enzymes, desired reaction selectivity can be programmed into the antibody by using an appropriately designed hapten. Catalytic antibodies almost always demonstrate a high degree of substrate selectivity.

In addition, catalytic antibodies have been produced that have regioselectivity sufficient to produce a single product for a reaction in which other products are normally observed in the absence of the antibody.

Finally, catalytic antibodies have been produced by immunization with a single-handed version (only left- or only right-handed) of a hapten, and only substrates with the same handedness can act as substrates for the resulting catalytic antibodies.

The net result is that a high degree of stereoselectivity is observed in the antibody-catalyzed reaction.

Abyzymes are artificial catalytic antibodies and come from the words “antibody” and “enzyme”

They are monoclonal antibodies that have catalytic properties, or carry out catalysis. The figure bellow shows the active of site of the abyzyme chorismate mutase and side-chain interactions with the transition state analog.

Specificity of the catalytic antibodies.

The substrate specificity of catalytic antibodies is generally excellent and reflects the power of immune recognition.

However, the effect of natural cocaine metabolites such as methyl leonine and nor-N-methyl ecgonine, whether as substrates or inhibitors must be assessed.

Also, ethyl cocaine has recently been identified as an active cocaine metabolite in those who co-abuse ethanol and cocaine.

An analog such as 3 would be very likely to elicit catalytic antibodies that would accept ethyl cocaine as a substrate.

However, the antibodies derived from analogs with the methyl ester of cocaine intact are loss predictable and we will synthesize ¹⁴ C-ethyl cocaine and study its hydrolysis by the artificial enzymes directly.