Surface enzymes

The data led to tire cycle shown in figure C2.7.8. Here, only tire active site on tire interior enzyme surface (section C2.6) is depicted, consisting of R groups including aspartic acid, glutamic acid and otliers, represented witli tire shortliand Asp, Glu etc tire subscripts represent tlie positions on tlie polypeptide chain. 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

Enzymes are protein molecules. While all enzymes are proteins, we do not imply that all proteins can act as enzymes. The protein molecules of enzymes are very large, with molecular weights of the order of 100,000. In contrast, the substance upon which the enzyme acts (called a substrate) is very small in comparison with the enzyme. This creates a picture of the reaction in which the small substrate molecule becomes attached to the surface of the large protein molecule, at which point the reaction occurs. The products of the reaction then dissociate from the enzyme surface and a new substrate molecule attaches to the enzyme and the reaction is repeated. We can write the following sequence ... 

Calibrating the Enzyme Surface Using Solution Experiments... 

In some cases, small biological redox partner proteins such as heme-containing cytochromes, ferredoxins comprising an iron-sulfur cluster, or azurin with a mononuclear Cu site have been used as natural mediators to facilitate fast electron exchange with enzymes. A specific surface site on the redox protein often complements a region on the enzyme surface, and enables selective docking with a short electron tunneling... 

Fig. 24. X-ray crystal structure of the catalytic domain of tyrosine hydroxylase. The catalytic iron is located 10 A below the enzyme surface and is coordinated by the conserved residues His-331, His-336, and Glu-376 (PDBID 1TOH). Adapted from (494). 

The initial steps in enzyme-catalysed reactions involve the binding of the reactants to the enzyme surface and one of the functions of the enzyme is to orientate these reactants relative to each other. This idea was suggested by Fischer as a lock-and-key hypothesis, where the enzyme is the lock and the... 

It seems that a maximum of about 10 ST-PHPMA chains can be attached to 1 CT molecule. The conjugation degree was lower than that of the chy-motrypsin conjugates with PEG-SC (succinimidyl carbonate of mPEG up to 14 chains per one CT molecule) [20,33]. This might be attributed to the solution stmcture difference between the two polymers. PHPMA has a random coil stmcture in aqueous solution, whereas PEG possesses a more extended one. The coiled PHPMA may cover more of the enzyme surface, and the steric effect may prevent more PHPMA macromolecules from attaching to the enzyme. 

Most cholinesterase inhibitors inhibit the enz)nne by acylating the esteratic site on the enzyme surface. Physostigmine and neostigmine are examples of... 

Irreversible anticholinesterases include the organophosphorus inhibitors and ambenonium, which irreversibly phosphorylate the esteratic site. Such drugs have few clinical uses but have been developed as insecticides and nerve gases. Besides blocking the muscarinic receptors with atropine sulphate in an attempt to reduce the toxic effects that result from an accumulation of acetylcholine, the only specific treatment for organopho-sphate poisoning would appear to be the administration of 2-pyridine aldoxime methiodide, which increases the rate of dissociation of the organophosphate from the esteratic site on the enzyme surface. 

Antidepressants of this class, such as moclobemide, have a high selectivity and affinity for MAO-A. Flowever, unlike the MAOIs, the RIMAs are reversible inhibitors of the enzyme and can easily be displaced from the enzyme surface by any primary amine which may be present in the diet. This means that the dietary amines are metabolized by MAO in the wall of the gastrointestinal tract while the enzyme in the brain and elsewhere remains inhibited. Thus the RIMAs have brought the MAOIs back into use as antidepressants in general practice. It is now evident that the RIMAs are not as potent as most currently available antidepressants. 

Most well-studied peroxidases are designed to oxidize small aromatic molecules, with the exception of cytochrome c peroxidase. It generally is thought that such aromatic molecules bind near the heme edge where an electron can transfer directly to the heme edge (44), which is supported by both crystal structures (45, 46) and NMR studies (47). However, recent work suggests that some physiologically important substrates may utilize other sites on the enzyme surface (48, 49). 

Katz, B. A. Finer-Moore, J. Mortezaei, R. Rich, D. H. Stroud, R. M. Episelection Novel K. approximately nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface. Biochemistry 1995, 34, 8264-8280. 

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