Trypsin substrate binding

Krieger, M., Kay, L.M., Stroud, R.M. Structure and specific binding of trypsin comparison of inhibited derivatives and a model for substrate binding. /. Mol. Biol. 83 209-230, 1974. 

FIGURE 16.19 The substrate-binding pockets of trypsin, chymotrypsin, and elastase. 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

Edges of P sheets can also serve as binding sites for other polar molecules. For example, substrates bind to an edge of a P sheet in the active sites of trypsin and other proteases (Chapter 12). Some proteins, e.g. the lectin shown in Fig. 2-15, form dimers by joining identical edges of a P sheet in antiparallel orientation.112... 

Several inhibitor-protease complexes have been crystallized and details of their interactions are known. For example, the pancreatic trypsin inhibitor binds at the active site of trypsin with K( >1013 M-1 at neutral pH 496 Tire two molecules fit snugly together,490 497 the inhibitor being bound as if it were a peptide substrate with one edge of the inhibitor molecule forming an antiparallel (1 structure with a peptide chain in the enzyme. Lysine 15, which forms part of this P structure, enters the specific Pj binding site for a basic amino acid in a substrate. Thus, the protease inhibitor is a modified substrate which may actually undergo attack at the active site. However, the fit between the two... 

The binding site for a polypeptide substrate consists of a series of subsites across the surface of the enzyme. By convention, they are labeled as in Figure 1.24. The substrate residues are called P (for peptide) the subsites, S. Except at the primary binding site S, for the side chains of the aromatic substrates of chymotrypsin or the basic amino acid substrates of trypsin, there is no obvious, well-defined cleft or groove for substrate binding. The subsites run along the surface of the protein. 

Fig. 11 Schematic representation of the molecular imprinting of trypsin using a polymerizable inhibitor as an anchoring monomer. The enzyme is put into contact with the anchoring monomer and co-monomers (a) polymerization is conducted (1) a cross-linked polymer is molded around the substrate binding site (b) the enzyme is removed (2), revealing a specific recognition site with inhibitory properties (c). Reproduced with permission from [108], Copyright 2009 American Chemical Society...

Fig. 2. Schematic representation of the substrate-binding sites in the serine proteases (a) trypsin, (b) chymotrypsin and (c) elastase.

M. Mares-Gtiia and E Shaw. Studies on the active center of trypsin. The binding of ami dines and guanidines as models of substrate side chain. J. BioL Chem. 240 1579 (1965),... 

The same lysyl residue 188 was replaced with histidine in order to build a metal chelation site in the substrate-binding pocket of trypsin (Briand et al., 1997). K188H mutation did not affect catalytic efficiency at all. In the presence of Cu2+, trypsin K188H exhibited a 30- to 100-fold increase of Km, while kcat was only slightly decreased (Table VII). Hydrolytic activity of this mutant could be fully restored by addition of EDTA. Thus, in contrast to the chelation of the active site, a different mode of metal-dependent inhibition of the activity of trypsin by building a co-ordination site in the substrate-binding pocket of the protease was achieved. 

Briand, L., Chobert, J.-M., Tauzin, J., Declerck, N., Leonil, J., Molle, D., Tran, V., and Haertle, T. 1997. Regulation of trypsin activity by Cu2+ chelation of the substrate binding site. Protein Engng 10, 551—560. 

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Serine proteases are widely distributed and have many different functions. They are products of at least two evolutionary pathways, which originate in prokaryotes. Many of them resemble trypsin, chymotrypsin, elastase, or sub-tilisin in specificity, but serine proteases with quite different specificities have been isolated recently. A recent NMR study of a bacterial protease labelled with at carbon 2 of its single imidazole groups implicates a buried side chain of aspartic acid as the ultimate base for proton transfers in catalysis and eliminates a charge separation from reaction schemes for catalysis. Much of the catalytic effectiveness of serine proteases can be attributed to substrate binding, but the interactions which yield a Michaelis complex are supplemented by others which stabilize intermediates on the reaction pathway. 

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