Chymotrypsin, complexation with

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.

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

The mechanism of action of anticholinesterases is to form a stable covalent complex with the Achase enzyme. Achase is one of several enzymes known as serine esterases. Other examples include the intestinal enzymes trypsin and chymotrypsin as well as the blood clotting agent thrombin. During the course of the catalysis the alcohol -OH of a serine side chain in the active site of the enzyme forms an ester complex, called the acyl-enzyme, with the substrate. So, acetylcholine will go through similar chemical reactions with Achase. 

Exercise 25-30 The proteolytic enzyme, papain, differs from chymotrypsin in having cysteine, or a labile derivative thereof, as part of its active site. The enzyme is deactivated by substances that form complexes with, or react with, —SH groups and the activity is restored by reactions expected to regenerate an —SH group. Work out a schematic mechanism for cleavage of a peptide chain with papain that involves acylation of the critical —SH group of papain. 

The overall reaction mechanism of chymotrypsin is sketched in figure 8.11. Part a shows the enzyme-substrate complex, with an aromatic side chain of the substrate seated in the binding pocket and the carbonyl oxygen atom hy-... 

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. 

Like antithrombin, heparin cofactor II inhibits proteases by forming a I I stoichiometric complex with the enzyme. The protease attacks the reactive site of heparin cofactor II located on the C-terminus, resulting in the formation of a covalent bond. Heparin cofactor II has higher protease specificity than antithrombin. Of the coagulation enzymes, heparin cofactor II is known only to inhibit thrombin (92). Additionally heparin cofactor II has been shown to inhibit chymotrypsin (93) and leukocyte cathepsin G (94), This protease specificity appears to be due to the active site bond present in heparin cofactor II. Whereas antithrombin contains an Arg-Ser bond as its active site, heparin cofactor II is unique in containing a Leu-Ser bond. This suggests than another portion of the heparin cofactor II molecular may be required for protease binding,... 

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... 

The mechanism of inhibition by benzoxazinones Figure 2.9) is believed to be similar [196, 197] to that of the alternate-substrate isocoumarins, and formation of a covalent acyl-enzyme complex with PPE has been confirmed by X-ray crystallographic studies [198], However, when is a hydrogen atom, deacylation of the acyl-enzyme via intramolecular ring closure can either reform the starting benzoxazinone (O attack) or lead to an isomeric quinazolinedione (N attack). It was shown for HLE and a-chymotrypsin that formation of the quinazolindione occurs faster than normal hydrolysis to the anthranilic acid. 

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). 

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