Antibody-directed catalysis

Figure 10.10). This approach has been termed antibody-directed enzyme prodrug therapy (ADEPT) or antibody-directed catalysis (ADC). 

The inhibitor endo-6-0 has had quite a career since we first described it in 1985. First, in the molecular equivalent of "turn around is fair play", it was used by Peter Schultz and David Jackson and by Don Hilvert and his coworkers to make catalytic antibodies.25-27 Rearrangement reactions in general, and especially those for which control of the substrate conformation is important, are particularly appropriate candidates for catalytic antibodies since they are unimolecular and seldom require covalent catalysis. The antibody obtained by Schultz and Jackson is reasonably efficient, accelerating the rearrangement 10,(X)0-fold at 0 C, which is 60% as good as the enzyme in stabilizing the transition state. It is interesting to compare the enzyme and the antibody directly, and... 

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

The topologically defined region(s) on an enzyme responsible for the binding of substrate(s), coenzymes, metal ions, and protons that directly participate in the chemical transformation catalyzed by an enzyme, ribo-zyme, or catalytic antibody. Active sites need not be part of the same protein subunit, and covalently bound intermediates may interact with several regions on different subunits of a multisubunit enzyme complex. See Lambda (A) Isomers of Metal Ion-Nucleotide Complexes Lock and Key Model of Enzyme Action Low-Barrier Hydrogen Bonds Role in Catalysis Yaga-Ozav /a Plot Yonetani-Theorell Plot Induced-Fit Model Allosteric Interaction... 

Similarly to their natural counterparts (enzymes, antibodies, and hormone receptors), MIPs have found numerous applications in various areas. They have been used as antibody mimics in immunoassays and sensors and biochips as affinity separation materials and for chemical and bioanalysis, for directed synthesis and enzyme-like catalysis, and for biomedical applications. Concerning their commercialization, there has been great progress during the past decade, in particular in the... 

A direct comparison of catalysis of olefin epoxidation with a homogeneous chemical catalyst (Mn salen), an enzyme (CPO), and an antibody resulted in sufficiently high enantioselectivity for all three catalysts, a higher turnover number for the enzyme, and a slightly higher substrate/catalyst ratio for the homogenous catalyst. Criteria for comparison should be quantitative and include catalyst lifetime as well as volumetric productivities, but have been found to depend on the different needs of laboratory synthetic chemists, who need a broadly specific catalyst quickly, versus those of process chemists, who often control catalyst availability and can allow narrow specificity (provided their substrate is accepted) but need high productivity. 

Control in biocatalytic systems comes from such new techniques as directed evolution [52], the formation of catalytic antibodies using transition state templates [53], and the powerful approach of site-directed mutagenesis [54], In both heterogeneous and enzymatic catalysis, rather than take what you get, we now try to design what we want and devise clever chemical and biological approaches to get it. 

The importance of catalysis in biological as well as synthetic organic chemistry cannot be overstated. In Chapter 2, Donald Hilvert examines the scope and utility of asymmetric reactions under catalysis by antibodies. From a stereochemical point of view, this has significant impact not only in the production of important compounds in stereochemically defined form, but also in the ability of the antibody catalysts to alter the stereochemical course of organic reactions in fashions contrary to their natural tendencies. The most important chemical transformations carried out by catalytic antibodies are covered and provide the reader with an excellent snapshot of the state of the art of this emerging subfield in asymmetric catalysis. In addition, a critical appraisal of the limitations and future directions is included which should provide ample stimulation for thought. 

The previous sections described structural studies of antibody maturation as a method to understand the evolution of binding (and catalysis) in the immune system. The technique of directed evolution parallels the process of affinity maturation. Both methods use random mutagenesis and gene shuffling, followed by screening and/or selection to identify mutants with the desired function. In contrast to affinity maturation,... 

III. Directing Evolution at the Level of Chemical Mechanism Aldolase Antibodies and Asymmetric Catalysis... 

The following new trends in enzymatic synthesis can be delineated the development of new enzymatic reactions enzyme immobilization and stabilization the use of organic solvents and two phase systems site-directed mutagenesis chemical modification of enzymes antibody catalysis catalysis by RNA and DNA de novo design ofbiocatalists employment of recombinant DNA for production of enzymes and use computational and combinatorial methods... 

It is very likely that nonaqueous solvents and mixtures can be of use in the elucidation of the mechanisms of biochemical reactions carried out by proteins, such as enzymatic catalysis and antigen-antibody interactions. With the rapidly accumulating knowledge of the effect of nonaqueous solvents on the conformation of proteins to guide us, it should be possible to carry out increasingly informative experiments in such directions. 

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