Chymotrypsin mechanism, hydrogen bonds

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...

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... 

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

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). 

Molecular mechanics minimization and molecular dynamics were chosen to examine the possible conformations for the two acyl enzymes and conclusions were drawn from the time evolution of the two systems. The starting point was a crystal structure of phenylethaneboronic acid bound to alpha-chymotrypsin. QUANTA/CHARMM (Brooks et al, 1983) was employed for the calculations. Ninety-five water molecules from the X-ray structure were included. Distance monitoring and the creation ofH-bonds were the main criteria for differentiating between the two molecules. Both acyl enzymes have their ketone carbonyls H-bonded to Gly-216 NH. Both start with their ester carbonyl in the oxyanion hole (H-bonded to Ser-195 and to Gly-193). The R-acyl enzyme looses both of these hydrogen bonds during the simulation. Attack of water on the R-species should, thus, be less frequently successful. Values for differences in energy were not used because of a small... 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

How do the critical amino acids catalyze the chymotrypsin reaction Nucleophilic attack by serine is the main feature of the mechanism, with histidine hydrogen-bonded to serine in the course of the reaction. The reaction takes place in two phases. In the first phase, serine is the nucleophile, and there is an acyl-enzyme intermediate. In the second phase, water acts as the nucleophile and the acyl-enzyme intermediate is hydrolyzed. 

Metabolites, in particular enzymes from bacteria and mold, attack the polymer skeleton, but more importantly the additives in the plastic material. Enzymes such as pepsin, trypsin, and chymotrypsin cleave the peptide bond in proteins and polyamides and can also hydrolyze ester bonds [32]. Their catalytic effect can activate hydrogen in the polymer chain, resulting in the formation of free radicals. The results of these processes are destroyed surfaces (Figure 5.371), loss of gloss, and changes in mechanical and electrical properties. 

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