Hydrogen bonding chymotrypsin

Fig. 5. Protein folding. The unfolded polypeptide chain coUapses and assembles to form simple stmctural motifs such as -sheets and a-hehces by nucleation-condensation mechanisms involving the formation of hydrogen bonds and van der Waal s interactions. Small proteins (eg, chymotrypsin inhibitor 2) attain their final (tertiary) stmcture in this way. Larger proteins and multiple protein assembhes aggregate by recognition and docking of multiple domains (eg, -barrels, a-helix bundles), often displaying positive cooperativity. Many noncovalent interactions, including hydrogen bonding, van der Waal s and electrostatic interactions, and the hydrophobic effect are exploited to create the final, compact protein assembly. Further stmctural...

Figure 11.9 A diagram of the active site of chymotrypsin with a bound inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH. The diagram illustrates how this inhibitor binds in relation to the catalytic triad, the strbstrate specificity pocket, the oxyanion hole and the nonspecific substrate binding region. The Inhibitor is ted. Hydrogen bonds between Inhibitor and enzyme are striped. (Adapted from M.N.G. James et al., /. Mol. Biol. 144 43-88, 1980.)...

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 /.

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. 

To realize the reason for this result from a simple intuitive point of view it is important to recognize that the ionized form of Aspc is more stable in the protein-active site than in water, due to its stabilization by three hydrogen bonds (Fig. 7.7). This point is clear from the fact that the observed pKa of the acid is around 3 in chymotrypsin, while it is around 4 in solution. As the stability of the negative charge on Aspc increases, the propensity for a proton transfer from Hisc to Aspc decreases. 

Fig. 54. An asparagine side chain making a hydrogen-bond to the main chain NH of residue n + 2, an arrangement which helps stabilize the central peptide of a tight turn. Residues 91-93 from chymotrypsin.

Chemical reactivity and hydrogen bonding 320 Proton-transfer behaviour 321 Intramolecular hydrogen-bond catalysis 344 Enzyme catalysis and hydrogen bonding 354 Chymotrypsin 354 Thermolysin 355 Carboxypeptidase 355 Tyrosyl tRNA synthetase 356 Summary 366 Acknowledgements 367 References 367... 

Numerous suggestions have been made that enzymes might owe part of their catalytic efficiency to the opportunity they afford for stabilization of intermediates or transition states by hydrogen bonding to functional groups near the active site. For example, in the case of (x-chymotrypsin this might be represented as in [43] where... 

The mechanism of action of chymotrypsin can be rationalized as follows (Figure 13.5). The enzyme-substrate complex forms, with the substrate being positioned correctly through hydrogen bonding and interaction with the pocket as described above. The nucleophilicity of a serine residue is only modest, but here it is improved by... 

More recently, Cassidy et aid conducted additional H NMR experiments to evaluate the basicities of the dyad H57-D102 in the tetrahedral complexes of chymotrypsin with the peptidyl trifluoromethyl ketones (TFKs) N-acetyl-L-Leu-DL-Phe-CFs and A-acetyl-oL-Phe-CFs. The proton bridging His-57 and Asp-102 is part of a low-barrier hydrogen bond (LBHBs). In H NMR spectra at pH 7.0, these protons appear at 8 18.9 and 18.6 ppm. 

Fig. 2. Model image of a typical substrate bound to ot-Chymotrypsin. (a) Binding of the substrate, (b) Three additional hydrogen bonds stabilize the intermediate oxyanion.

The strongest influence of configuration has been observed for Pi -substituted diastereomers of Z-Phe-(aTfm)Ala-Ala-NH2. The crystal structures of both dia-stereomers have been solved, which enables a better interpretation of this rather interesting effect. While the (S,S,S)-diastereomer has been shown to be almost as stable as the Aib-substituted peptide, the (S,R,S)-diastereomer was hydrolyzed very quickly within the same time range. Molecular modeling studies readily support the formation of hydrogen bonds as a possible explanation for this effect [18,54]. With the known crystal structure of the a-chymotrypsin/phenyl boronic... 

Support for this concept is provided by H NMR studies which have identified a downfield resonance of the hydrogen-bonded proton in this pair at 18 ppm in chymotrypsinogen and chymotrypsin at low pH and at 14.9-15.5 ppm at high pH values.246 247 Similar resonances are seen in the a-lytic protease,248 in sub-tilisin,249 in adducts of serine proteases with boronic acids250 251 or peptidyl trifluoromethyl ketones,252 in alkylated derivative of the active site histidine,253 and in molecular complexes that mimic the Asp-His pair in the active sites of serine proteases.254... 

Trypsin 66,116, 609. See also Chymotrypsin hydrogen-bonding network, structure 612... 

Figure 1.18 Comparison of the binding pockets in (a) chymotrypsin, with IV-formyl-L-tryptophan bound, and (b) elastase, with IV-formyl-L-alanine bound. The binding pocket in trypsin is very similar to that in chymotrypsin, except that residue 189 is an aspartate to bind positively charged side chains. Note the hydrogen bonds between the substrate and the backbone of the enzyme.

The resonances of protons in hydrogen bonds may be shifted downfield to such an extent that they may be observed in H20 solutions. The proton between Asp-102 and His-57 in chymotrypsinogen, chymotrypsin, and other serine proteases has been located and its resonance found to titrate with a pKa of 7.518 (although the pKa is for the dissociation of the proton on the other nitnjjgen of the imidazole ring). ... 

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