Chymotrypsin: An Enzyme at Work
The principles of enzyme action are illustrated by the enzyme chymotrypsin. Chymotrypsin digests proteins in the intestine by hydrolyzing the peptide bond at the carboxy side (to the right as conventionally written) of a hydrophobic amino acid. Thus, the small peptide glycylphenylalanylglycine (GlyPheGly) is hydrolyzed to GlyPhe and Gly.
The active site of chymotrypsin contains several reactive groups in close proximity to the binding site for the hydrophobic amino acid side chain. This binding site is a deep pocket lined with hydrophobic amino acid side chains. A charged amino acid side chain has to give up its favorable interactions with water to insert into the binding pocket, but the hydrophobic side chain of phenylalanine, for example, gains a favorable interaction and leaves water behind. When the substrate is bound to the enzyme, nearby amino acid side chains of the active site participate in the enzymatic reaction. Figure 1 shows the process inside such a pocket.
Three amino acid side chains participate in the catalytic reaction by forming a charge relay system. One amino acid, serine, is partially deprotonated by a nearby histidine. Normally, histidine is not a strong enough base to remove a proton from serine—but the histidine is itself partially deprotonated by the carboxylate side chain of an aspartate. The end result of this charge relay system is that serine is able to attack the carbonyl carbon, breaking the peptide bond and forming an acyl enzyme intermediate. The proton originally bound to the serine hydroxyl group is transferred to the amino group in the peptide bond, leaving histidine able to accept a proton from water. The rest of the water attacks the acyl enzyme intermediate, leading to the reforming of the original enzyme.
The importance of these amino acid side chains is illustrated by the action of two kinds of irreversible enzyme inhibitors (shown in Figure ). Diisopropylfluorophosphate transfers its phosphate to the active site serine. The resulting phospho‐enzyme is totally inactive. Chloromethyl ketones alkylate the active site histidine.
Enzyme inhibitors are often poisonous. For example, diisopropylfluorophosphate is a nerve poison because the enzyme acetylcholinesterase has a reactive site serine. Chymotrypsin and acetylcholinesterase are both members of the class of enzymes known as serine esterases, which are all inhibited by diisopropylfluorophosphate.