Chymotrypsin, complexation with inhibitors

The oxyanion hole geometry of three complexes is visuahzed in Figures 4.2. 4. Figure 4.2 displays the active site of trypsin complexed with a peptide inhibitor [41]. In Figure 4.3, the active site of chymotrypsin complexed with a neutral aldehyde adduct is displayed [43], and in Figure 4.4, cutinase (a lipase) with a covalently bound phosphate, a transition state analog is depicted [63]. 

Figure 4.3 Structure of the oxyanion hole of the active site of chymotrypsin complexed with a peptide inhibitor (PDB ICCD) (the same view as used in Figure 4.2). As discussed in the text, it is believed [43] that the oxygen atom in the oxyanion hole is protonated and therefore neutral.

Evidence for the tetrahedral intermediate includes a Hammett p constant of+2.1 for the deacylation reaction of substituted benzoyl-chymotrypsins and the formation of tetrahedral complexes with many inhibitors, such as boronates, sulfonyl fluorides, peptide aldehydes, and peptidyl trifluoromethyl ketones. In these last the chemical shift of the imidazole proton is 18.9 ppm, indicating a good low-barrier H-bond, and the pJQ of the imidazolium is 12.1, indicating that it is stabilized by 7.3 kcal mol 1 compared to substrate-free chymotrypsin. The imidazole in effect is a much stronger base, facilitating proton removal from the serine. 

The fold of the serine protease domain-type was described in Section V.C. SCP is more like o-lytic protease (Fujinaga et al, 1985) than a-chymotrypsin (Matthews et al, 1967), but with loops that are even shorter (Fig. 4 see Color Insert). Unlike the other proteases, there are no disulfide bonds. The structure of the C terminus is completely different from either of the other two proteases, and it leaves the final three amino acids in the active site. These superimpose on the structure of a peptide inhibitor determined as a complex with o-lytic protease (Bone et al, 1987). This indicates that the N-terminal product of the autocatalytic lysis of... 

Two cases were studied by Crippen (268). The first was a set of eight phenoxyacetone inhibitors of chymotrypsin. A set of site points was developed that rationalized the experimental binding data. Although the X-ray crystal structure of a-chymotrypsin was known, no structures of a complex with the phenoxyacetone inhibitors were available and therefore a direct comparison of Crippen s site points with the X-ray structure was not possible. It was possible, however, to fit these site points and the ligand into the chymotrypsin X-ray coordinates and show they were compatible. 

M. G. GrQtter, G. Fendrich, R. Huber, and W. Bode. The 2.5 A X-ray crystal structure of the acid-stable proteinase inhibitor from human mucous secretions analysed in its complex with bovine a-chymotrypsin. EMBO J. 7 345 (1988). 

It appears, therefore, that trypsin and chymotrypsin recognize and bind with the same amino acid residues in the inhibitors as with any substrate. However, net hydrolysis of peptide bonds does not occur at the pH optimum of the enzymes for reasons largely unknown at this time. Specific hydrolysis of the peptide bond involving the carboxyl group of the essential amino acid residue does occur at low pH (around pH 4). However, there is little current evidence to indicate that this hydrolysis is a key step in the inhibitory process. Two types of data argue against its essentiality, (a) Complexation between inhibitor and chemically modified Inactive proteases is often just as tight as with the native protease (12, 100, 101). (b) Initially, X-ray... 

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... 

Soybean trypsin inhibitor, STI the best known plant trypsin inhibitor. AMth bovine trypsin, at pH 8.3, it forms a stoichiometric, enzymatically inactive, stable complex with an association constant of 5 X 10 per mol STI. It also inhibits other vertebrate and invertebrate trypsins and plasmin. Chymotrypsin is inhibited to a small extent, and other endopeptida-ses not at all. STI is a single polypeptide chain, M, 21,100,181 amino acid residues (of known sequence) and two disulfide bridges. The molecule is compact and has a low a-helix content due to the presence of proline residues. It is consequently resistant to proteases and to denaturation. The reactive center contains a specific peptide bond Argj3-Ile(4, which is hydrolysed when the trypsin-STI complex is formed. The ensuing interaction with the active site of trypsin. 

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). 

Blrk family (Laskowski and Kato, 1980) are small proteins with disulfide crosslinks between the proteinase-binding regions. Bound trypsin or chymotrypsin was stabilized In Its 1 1 complex with LBI both enzymes were stabilized In the ternary (trypsin—LBI—chymotrypsin) complex (Fig. 7). With highly stable Inhibitors, such as LBI or BPTI, association produced no observable stabilization of the Inhibitor (Zahnley, 1979,1980). 

Each of the multiheaded Inhibitors described above Is a monomer, with one enzyme-binding region per covalently linked domain. Effects of association of protelnases with multimeric Inhibitors, which have the enzyme-binding regions on separate subunits (noncovalently associated), on stability have also been measured by DSC. Considerable stabilization of chymotrypsin and subtil 1 sin was observed In complexes with proteinase Inhibitors I and II from potato (Table III). Chymotrypsin showed Increases In Td of 33 C (major peak) on binding to... 

Fujinaga, M., et al. Crystal and molecular structures of the complex of a-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. 

Serine protease inhibitor that inhibits trypsin, chymotrypsin, kallikrein, and plasmin Binding is reversible, with most aprotinin-protease complexes dissociating at pH>10or <3 Peptidase inhibitor... 

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 ... 

Experimental support for the above mechanistic interpretation comes from the work of Bizzozero and Zweifel (9) who have studied the behavior of N-acetyl-j -phenyl al anyl- -prol i ne amide ( ) and N-acetyl-L-phenylalanyl-sarcosine amide (32) toward enzymic hydrolysis with o-chymotrypsin. These two dipeptides were found to be good competitive inhibitors with a specific substrate (Ac-Phe-0CH3 (33)) but no hydrolysis was observed. These two peptides thus form an enzyme-substrate complex and the reason for their nonreactivity has to be sought in the nature of the enzyme-substrate interactions occurring during the subsequent bond-change steps. 

Like SLPI, elafin is a very potent inhibitor of NE. Elafin interacts with elastase in a reversible manner and retains its inhibitory capacity upon dissociation of the complex. Kinetic data have shown that the association rate constant for the formation of an elafin-elastase complex is pH dependent [84]. Una is probably due to the bask properties of the inhibitor and enzyme. Elafin also inhibits proteinase 3 but has no effect on cathepsin G, trypsin, or chymotrypsin. This is inloestiitg in view of the fact that SLPI inhibits cathepsin G but la relatively ineffective against proteinase 3. 

Fig. 19. Stereodrawing of 6-benzyl-3-chloro-2-pyrone bound to the active site of y-chymotrypsin. The structures of the native enzyme (solid lines) and the enzyme-inhibitor complex (clear lines) are overlaid. Reproduced with permission from Ringe et al. (1985).

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