Serine protease binding groups

Figure 3.3 (a) Covalent catalysis the catalytic mechanism of a serine protease. The enzyme acetylcholinesterase is chosen to illustrate the mechanism because it is an important enzyme in the nervous system. Catalysis occurs in three stages (i) binding of acetyl choline (ii) release of choline (iii) hydrolysis of acetyl group from the enzyme to produce acetate, (b) Mechanism of inhibition of serine proteases by diisopropylfluorophosphonate. See text for details. 

To summarize, the binding sites of lysozyme and the serine proteases are approximately complementary in structure to the structures of the substrates the nonpolar parts of the substrate match up with nonpolar side chains of the amino acids the hydrogen-bonding sites on the substrate bind to the backbone NH and CO groups of the protein and, for lysozyme, to the polar side chains of amino acids. The reactive part of the substrate is firmly held by this binding next to acidic, basic, or nucleophilic groups on the enzyme. 

Finally, three groups have reported the preparation of artificial enzymes with catalytic activity. Stewart and co-workers [73] incorporated a catalytic triad from the serine proteases into a designed four a-helix protein (80). In their design, they incorporated one of the amino acids involved in the catalytic function at the N-terminal side of the a-helices that are linked together by their C-terminal position (Fig. 29). The authors proposed that the oxyanion hole and the hydrophobic binding pocket are created by the three-dimensional structure formed by the folding of 80. Compared to the spontaneous reaction, impressive... 

Several pharmaceutical enzymes belong to the group of serine-histidine estero-proteolytic enzymes (serine proteases), which display their catalytic activity with the aid of an especially reactive serine residue, whoso p-hydroxyi group forms a covalent bond with the substrate molecule. This reaction takes place by cooperation with the imidazole base of histidine. The specificity of the enzymes is achieved by the characteristic strocture of their substrate-binding centers, which in these proteases are built according to the same principle. They consist of a hydrophobic slit formed by apolar aide chains of amino acids and a dissociated side chain-located carboxyl group of an aspartic add residue at the bottom. 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

A good example of this is the classic work by Bender (6) on the reaction ofra-f-butylphenyl acetate. This substrate binds well into the cavity, and the substrate then undergoes an acetyl transfer reaction in which a cyclodextrin hydroxyl group is acetylated. The reaction can be compared with the first step in the action of a serine esterase, or a serine protease acting on an ester substrate. However, the acceleration of this acetyl transfer, compared with simple hydrolysis by the medium, was only 250-fold. 

The second group of approaches, seeking similarities in the shapes and chemical surface properties of binding sites, include recently developed tools such as CavBase [89, 90], eF-Site [91], and SuMo [92]. An earlier technique using surface shape only for binding site comparisons was reported by Rosen et al. [93], The reliability of this geometric surface-matching approach has been shown for the catalytic triad of serine proteases and chorismate mutase. 

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