Subtilisin-chymotrypsin inhibitor

Figure 7.11 Linear free energy correlation plots for inhibition of subtilisin BPN mutants by wild type (open circles) and mutant (close circles) chymotrypsin inhibitor 2. Left panel Correlation between AGbinding for the inhibitor and AGm. Right panel Correlation between AGbinding for the inhibitor and AGES.

A large number of potential reversible protease inhibitors exist (Laskowski Kato, 1980). Protein protease inhibitors like Strepromyces Subtilisin Inhibitor (SSI) (Hiromi et al, 1985) and Chymotrypsin Inhibitor (CI-2) (Jonassen, 1980 and McPhalen James, 1988) are known to be very strong inhibitors with inhibition constants at or below 10"10 M. 

Despite their lack of stabilizing disulfide bridges Potl inhibitors feature a common, stable fold. The N-terminus is coiled, although in some structures a small /3-strand has been identified. After a turn the structure adopts an a-helical structure, followed by a turn and an other /3-strand. The sequence then features an extended turn or loop motif that contains the reactive site of the inhibitor before it proceeds with a /3-strand running almost parallel to the /3-strand after the a-helix. After another turn and coiled motif a short /3-strand antiparallel to the other /3-strands precedes the coiled C-terminus. Usually the N-terminal residue in the reactive site is an acidic residue followed by an aromatic amino acid, that is, tyrosine or phenylalanine. Figure 11 shows the complex of chymotrypsin inhibitor (Cl) 2 with subtilisin, the hexamer of Cl 2 from H. vulgare and a structural comparison with a trypsin inhibitor from Linum usitatissimum ... 

Results of protein-protein docking. aCHYN-a-chymotrypsinogen, a-CHY-a-chymotrypsin, HPTI-human pancreatic trypsin inhibitor, BPTI-bovine pancreatic trypsin inhibitor, CHYI-chymotrypsin inhibitor, Subtilisin l-subtilisin inhibitor D1.3, D44.1, HyHEL5 and HyHELlOare monoclonal antibodies. For further details of coordinates see Cabb et al. [12]. In the table some degenerate identical complexes included our earlier studies have been excluded. 

Figure 11.14 Schematic diagram of the active site of subtilisin. A region (residues 42-45) of a bound polypeptide inhibitor, eglin, is shown in red. The four essential features of the active site— the catalytic triad, the oxyanion hole, the specificity pocket, and the region for nonspecific binding of substrate—are highlighted in yellow. Important hydrogen bonds between enzyme and inhibitor are striped. This figure should be compared to Figure 11.9, which shows the same features for chymotrypsin. (Adapted from W. Bode et al., EMBO /.

CI2 (Figure 19.2) is a 64-residue polypeptide inhibitor of serine proteases.23 It has a binding loop (Met-40, which binds in the primary site of chymotrypsin or subtilisin), a single a helix running from residues 12 to 24, and a mixed parallel and antiparallel /3 sheet. The strands and the amphipathic helix interact to form... 

Of particular interest is the study of the biological mechanisms associated with enzyme stereoselectivity and enantioselectivity. For example, MD simulations have been successful in explaining the different affinities of trypsin and acetylcholinesterase to the diastereomers of soman inhibitors [154] and the ability of subtilisin Carlsberg and a-chymotrypsin to discriminate between R-and S- configurations of chiral aldehyde inhibitors [155, 156]. 

Both Ser 195 and His 57 (or corresponding residues) are present in the active site (Figs. 12-9,12-10). From the observed positions of competitive inhibitors occupying the active site, the modes of binding depicted in Fig. 12-lOA for the chymotrypsin family and in Fig. 12-lOB for the subtilisin family have been deduced. Bear in mind that the X-ray diffraction results do not... 

Chymotrypsin and subtilisin probably bind at the same site on these inhibitors not determined. 

Previous studies have demonstrated that subtilisin has a chymotrypsin-like specificity (reviewed in 9 ), preferring to hydrolyze the peptide bond following a large hydrophobic residue. A model of substrate binding has been deduced from the three-dimensional models of peptide and protein inhibitor complexes with subtilisin (10). The model we have derived is shown in Figure 1. The enzyme can be divided in a series of subsites... 

Such effects are not unique to carboxypeptidase A. The rate of substrate polysaccharide hydrolysis by lysozyme is remarkably dependent on the polysaccharide chain length 153, 154). Both steady-state kinetic studies and X-ray crystallographic studies on enzyme-inhibitor complexes for chymotrypsin 156) trypsin 156), elastase 157), and subtilisin 158) are indicative of the existence of multiple-loci substrate binding sites. Furthermore, the dependence of Acat on substrate chain length for all these enzymes strongly implies that the filling... 

The studies now reported show that chloromethyl ketone polypeptide inhibitors bind in an antiparallel jS-pIeated sheet fashion to a length of extended backbone, Ser-125—Leu-126—Gly-127 in subtilisin, and Ser-214— Trp 215—Gly 216 in y-chymotrypsin. In each case there are the same geometric relationships of the pleated sheet to the active serine, and glycine residues are involved in j3-pleated sheet hydrogen bonding in both (see Figure 2). In the case of y-chymotrypsin these deductions were made from... 

The pancreatic trypsin inhibitor binds to trypsin, chymotrypsin, plasmin, and kallikrein, but does not inhibit elastase and subtilisin. Model-building studies of the inhibitor chymotrypsin complex show that the enzyme and Inhibitor have highly complementary structures. If Lys-15 (in a non-protonated form) is placed in the spedficity pocket with the C and NH of Lys-15 in similar positions to those of tryptophan in the formyl-L-tryptophan-chymotrypsin complex, the residues on the AT-terminal side of lysine then form an antiparallel jS-structure with the enzyme similar to that proposed for y-chymostrypsin (1). There appear to be a number of favourable hydro-... 

The relative affinities (Osuga et al., 1974) allow subtilisin to displace chymotrypsin from the complex with ovoinhibitor. Excess chymotrypsin and inhibitor (mole ratio 3 1) were allowed to react for 6 min, then one mole of subtilisin per mole of inhibitor was added and the mixture was heated in the DSC (Zahnley, 1980). The thermogram (Fig. 6) lacks the chymotrypsin—ovoinhibitor (2 1) peak at 72 C and the free subtilisin peak at 84 C, but it shows peaks coinciding with those for the 1 1 complexes (dashed lines) and the mixed chymotrypsin—ovoinhibitor—subtilisin complex (top thermogram). 

The specificity of compound 1 for AChE was demonstrated by treating other proteases of the His-Ser-Asp charge relay mechanism with the potential inhibitor. Trypsin, elastase, and subtilisin were not inactivated by 1. Chymotrypsin and kallikrein showed barely detectable inactivation at 100 IM and 40-45% inactivation at 1 mM. Conpounds 2, 3, and 5 also inactivated the eel AChE. Compound 2 showed 95% inactivation at 100 IM in 15 min. Conpounds 3 and 5 showed IC50 values of 102 XM and 108 1M, respectively. 

The very first examples were some simple boronic acid compoimds [5, 6]. For example, phenylboronic acid and substituted phenylboronic acids were found to be strong competitive inhibitors of subtilisin and chymotrypsin [6]. More recendy, boronic acids have been used for the synthesis of inhibitors against thrombin [50,51], lactamases [52], dipeptidyl peptidases [53], and others [54, 55]. In this section, thrombin inhibitors will be used as examples. The rest will be summarized in a table at the end of the section. 

In addition to the trypsin inhibitors are very interesting inhibitors of chymotrypsin and subtilisin, which are the most heterogeneous structure and variability of all species of wheat and corn and barley, which are controlled by fifth chromosome, while the wheat and rye - I homoeologous group of chromosomes of different genomes. In diploid wheat, a number of species (T, timopheevii) identified the protein components capable of inhibiting both subtilisin and chymotrypsin, which are characterized by low variability (Konarev et al., 2004). 

Fig. 3.13. Proportionality of the loss of accessible surface area to the molecular weight of proteins (from Janin, 1976). Different proteins insulin, rubredoxin, pancreatic trypsin inhibitor, HIPIP, calcium binding protein, ribonuclease S, lysozyme, staphylococcal nuclease, papain, chymotrypsin, concanavalin A, subtilisin, thermolysin, carboxypeptidase A.

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