Chymotrypsin hydrogen bond stability

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.

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.

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... 

Fig. 6 Mechanism of chymotrypsin.5 A low-barrier hydrogen bond between Aspl02 and His57 helps stabilize the tetrahedral intermediate.

Figure 9.9. The Oxyauion Hole. The structure stabilizes the tetrahedral intermediate of the chymotrypsin reaction. Hydrogen bonds (shown in green) link peptide NH groups and the negatively charged oxygen.

Circiunstantial support for this mechanism was supplied by the fact that A-tosyl-Phe-CMK, a specific inhibitor of chymotrypsin, did not react with anhydrochymotrypsin [104]. Although both X-ray crystallographic and NMR studies supported the alkylated hemiketal as the structure of the inhibited enzyme, those studies did not prove whether alkylation or hemiketal formation oecurred first [105, 98]. Carbon-13 NMR studies were also used to determine the pKa (7.88-8.1) of the hemiketal hydroxyl and this finding provided the first evidence that serine proteinases could stabilize the ionized form of the alkylated hemiketal, via hydrogen bonds in the oxyanion hole [106,107]. A series of more recent papers has confirmed that hemiketal formation precedes the alkylation step and has shown that the initial, reversible part of the interaction is made up of two discrete stages (a) formation of a Michaelis complex, followed by (b) hemiketal formation [102, 108]. The requirement of an intermediate hemiketal may mean that chloromethyl ketone (CMK) inhibitors should be considered as transition-state [109] analogue inhibitors (see diseussion in seetion on Aldehydes). 

X-ray structures have been worked out for the benzeneboronic and 2-phenyl-ethaneboronic acid (PEBA) complexes of subtilisin [9] and for the PEBA complex of a-chymotrypsin (a-CHT) dimer [10]. Further stabilization of the hydroxyls on boron is gained by hydrogen bonding to other amido groups lining the oxyanion hole. 

The catalytic efficiency of a-chymotrypsin cannot be solely attributed to the presence of the charge-relay system. X-Ray work (81) has indicated the many parameters operative in the catalytic process. Nine specific enzyme substrate interactions have been identified in making the process more efficient. For example, stabilization of the tetrahedral intermediate, and thus lowering of the transition state energy barrier, is accomplished by hydrogen bond formation of the substrate carbonyl function with the amide hydrogen... 

In RmL, 081 of Asp-203 accepts a syn-type H-bond from N81 of His-257 and an anti-type bond from Or/ of Tyr-260 (Fig. 5A). On the other hand, 082 is within a small distance of both N81 of His-257 (3.02 A) and the main-chain N of Val-205 (2.97 A). However, in the former case the angle on the hydrogen atom (082 H—Ne2) is much too acute ( 80°) for a strong H-bond. It is therefore the interaction with Val-205 that is relevant with respect to the stabilization of the side-chain conformation of Asp-203. This stereochemistry is very close to that found in chymotrypsin (Fig. 5B), where 081 of Asp-102 also accepts two H-bonds a syn type from Ne2 of His-57 and an anti from the hydroxyl of Ser-114. The overall effect is such that in lipases and in chymotrypsin the carboxyl group of the catalytic Asp is oriented in the same way with respect to the plane of the imidazole of the catalytic histidine. 

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