Polypeptide substrate with binding site

Other potent peptide mimetic NS3 protease inhibitors have been reported that incorporate a serine trap on the C-terminal end of the peptide. Thus, the inhibitory activity of telaprevir (VX-950, 59), (7nM vs. NS3, 300 nM vs. the la replicon) is based on truncation of the polypeptide substrate, maximizing binding by alteration of amino acids at the scissile site, and capping both N- and C-terminal ends, the latter with a known dicarbonyl serine trap. This compound has exhibited impressive antiviral activity in animals, and showed a 4.4 log drop in viral load in genotype 1-infected patients in a Phase lb clinical trial [110]. Telaprevir is expected to enter Phase 3 clinical trials in 2007. Additional bicyclo-proline-based P2 tetrapeptides, represented by analog 60 (Kj = 22 nM), have been explored. Although the compounds are selective inhibitors of NS3, little or no cell-based replicon activity was reported, presumably due to poor cellular permeability [111-114], A diastereomer of telaprevir, has been reported to inhibit the replicon with an EC50 of 0.55 pM [115]. 

Figure 2. A representation of the interaction of a polypeptide substrate with the extended substrate binding site of a protease. The individual amino acid (AA) residues are designated Pl7 P2, P/ etc. and the corresponding subsites of the enzyme are Sl7 S2, S/ etc. The bond cleaved by the enzyme is the peptide bond between the Pt and P/ residues.

Figure 1. A representation of the interaction of polypeptide substrate with the extended substrate binding site of pepsin.

Citrate synthase from mitochondria has been crystallized and visualized by x-ray diffraction in the presence and absence of its substrates and inhibitors (Fig. 16-8). Each subunit of the homodimeric enzyme is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the active site between them. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational... 

Active sites of enzymes and binding sites of proteins are a general source of instability because they contain groups that are exposed to solvent in order to bind substrates and ligands and so are not paired with their normal types of partners. Stability-activity trade-off is also seen with residues in the natural polypeptide inhibitor of barnase, barstar, that has evolved to bind as rapidly as possible to barnase,70 and also in the active site of T4 lysozyme.71... 

Residues in the region of the substrate-binding site for phosphate in phosphorylase b in the T state (a) and the R state (b). The side chain of Asp 283 leaves the binding site in the R structure, and the side chain of Arg 569 becomes available to interact with the phosphate. Key side chains and bound phosphate anion are shown in red. Portions of the polypeptide backbone are drawn in heavy black. (Source From D. Barford and L. N. Johnson, The allosteric transition of glycogen phosphorylase, Nature 340 609, 1989.)... 

The iron protein has been found in animals, viruses and some prokaryotic organisms. The best studied example is from Escherichia coli.S19 The enzyme consists of two subunits, Bi and B2. Subunit B, has a molecular weight of 160 000 and is made up of two polypeptide chains. It contains redox-active SH groups, which play a role in the reaction, and has two binding sites for substrates and sites for effector molecules. It can bind any of the four ribonucleotide substrates. Subunit B2 has a molecular weight of 78 000 and also has two polypeptide chains. It contains a dimeric oxo-bridged iron site associated with a remarkably stable tyrosine radical. The formation of the active enzyme from B, and B2 requires the presence of magnesium ions. 

The active sites of proteinases not only contain the catalytic site but also specific binding sites on the enzyme surface, which interact noncovalently with individual amino add residues of the polypeptide substrate, The peptide bond hvdrolvzed bv the nroteinase is called the sdssile or Ihe Pl-Pl bond... 

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