Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Enzymes
Catalytic antibodies ("abzymes")

The creation of artificial ENZYMES, known as abzymes, served to verify our understanding of the principles of Enzymatic Catalysis—specifically, the crucial role played by the stabilization of the transition complex through its selective binding at the catalytic site. Abzymes (derived from AntiBody + enZYME) are Antibodies that specifically bind structures chemically similar to the Transition State formed during catalysis. The method for generating abzymes is based on the fact that among the vast repertoire of possible immunoglobulin structures (see Chapter 13), one can always find those that contain appropriately oriented groups and are capable of selectively binding stable chemical compounds structurally analogous to the transition state of a given reaction.

When a complex is formed with such an immunoglobulin, favorable conditions arise for the substrate's conformation to approximate that of the transition state. In other words, the activation energy of the substrate is lowered—that is, the energy required to drive it from the ground state into the transition state. Consequently, the probability of its conversion into a product increases, and along with it, the reaction rate. Naturally, this is not yet about creating catalysts equivalent in efficiency to natural enzymes, but rather about obtaining their simplified models, although some of these may well prove practically useful.

The initial approach to designing abzymes involved generating antibodies that specifically bind and stabilize transition-state analogs characteristic of hydrolytic reactions. Obviously, an antibody directed against a compound (hapten) that mimics the configuration of the transition state will stabilize this Structure and thereby lower the Free energy of activation. It is well established (see Section 10.5) that the Hydrolysis of amides and esters (for example, by alkali) proceeds via The formation of a tetrahedral intermediate:

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Analysis of the MECHANISM OF ACTION of hydrolytic enzymes, particularly Serine proteinases (see Section 10.5), demonstrates that a key feature of their action is indeed the stabilization of a transition state structurally similar to such a tetrahedral intermediate. Although it is impossible to synthesize the corresponding compound that would fully mimic this structure—containing three oxygen atoms (or two oxygen atoms and one nitrogen atom in the case of proteinase modeling) attached to a single carbon atom—because it is far too unstable, it is nevertheless possible to obtain sufficiently stable tetrahedral stereochemical analogs in which a phosphorus atom occupies the central position instead of a carbon atom. Coupling such compounds to a carrier protein as haptens and immunizing animals with these derivatives makes it possible to obtain antibodies that specifically bind molecules whose structure and conformation closely resemble the transition state through which the hydrolysis of carboxylic acid esters or amides occurs.

Thus, in developing abzymes designed to accelerate the hydrolysis of the carbonic acid ester *n*-nitrophenyl *N,N,N*-trimethylammoniumethyl carbonate

— IMMUNOGLOBULINS were obtained that specifically interact with the tetrahedral charged phosphonate and phosphate analogs of the transition state of this compound's hydrolysis reaction, which has the following structure:

Here

Its model—the phosphonate analog—

possesses a structure that is stereochemically similar, albeit differing in several details—specifically, the direct attachment of the side-chain carbon to the central phosphorus atom, the length of this chain, the density of negative charges on the oxygen atoms, and so forth. The phosphonate analog was coupled via a carboxyl group to a carrier protein. Monoclonal Antibodies raised against this construct were found to catalyze the hydrolysis of the substrate, the carbonic acid ester:

The kinetics of this reaction, like that of conventional enzymatic reactions, obey the Michaelis–Menten Equation. Hydrolysis is inhibited by transition-state analog compounds, with The values of K_i being significantly lower than the substrate's K_m. Consequently, as expected, the antibody binds the transition state analog (reflected by K_i) more tightly than the substrate (characterized by K_m). This confirms the critical importance of transition-state

stabilization for catalysis. The reaction rate depends on the first power of the hydroxyl ion concentration and exceeds the non-catalyzed rate by a factor of 10³–10⁴. Naturally, an enzymatic reaction would yield a much greater acceleration by several orders of magnitude; however, it must be taken into account that the abzyme design modeled only one, albeit very significant, feature of enzymatic catalysis—the stabilization of the transition state. Furthermore, as noted, the binding of the phosphonate analog only approximates rather than perfectly reproduces the network of stabilizing interactions. The fundamental result remains significant: the creation of a catalyst, however insufficiently efficient as yet, based on our current understanding of the core Features of the catalytic process.

The reaction exhibits strict substrate Specificity: the antibody against the analog catalyzes the hydrolysis only of the aforementioned compound, but not of the related methyl-*n*-nitrophenyl carbonate

X-ray crystallographic analysis of an antibody that binds phosphorylcholine—which likely shares structural features with the catalytic antibody under consideration—reveals that the guanidino group of the Arg-52 residue and the hydroxyl group of Tyr-33 in the heavy chain can interact with the oxygen of the phosphate group. It is probable that in the catalytic antibody, the transition state is stabilized in a similar manner, with these same amino acid residues engaging the oxygen atoms of its structure. At the same time, the Glu-35 residue, also located in the heavy chain, forms an ionic bond with the ammonium group of the substrate, thereby contributing to the abzyme's specificity.

Another approach to modeling the principles of enzymatic catalysis involves utilizing antibodies to properly bring reacting components or parts of a transforming molecule into close proximity within the Active Site. For example, an antibody that specifically binds a bicyclic inhibitor of chorismate mutase selectively accelerates The conversion of Chorismic acid into prephenic acid by a factor of 10⁴ (k_cat = 2.7 min^-1, K_m = 260 µM).

An antibody raised against the phosphate analog of a 5-lactone, whose formula is given below, accelerates the cyclization of the phenyl ester of the corresponding δ-hydroxy acid into a lactone by a factor of 167:

The Introduction of additional functional groups into catalytic antibodies also opens up exciting possibilities. Thus, the generation of abzymes not only confirms the validity of modern concepts regarding The Nature of enzymatic catalysis but also paves the way for the creation of novel biological catalysts, including those for reactions that do not exist in nature.