Chymotrypsin is a digestive enzyme, classified as a serine protease, that breaks down proteins in the small intestine. This enzyme helps the body process dietary proteins, allowing their amino acids to be absorbed and utilized.
Building Blocks of Chymotrypsin
Chymotrypsin’s foundation is its primary structure, a linear sequence of amino acids. The bovine form, a commonly studied example, is initially synthesized as a single polypeptide chain of 245 amino acids. This sequence dictates how the protein will fold into its complex three-dimensional shape, ultimately determining its function.
Within this linear chain, localized folding events give rise to secondary structures: alpha-helices and beta-sheets. Alpha-helices are spiral-shaped segments, while beta-sheets are flattened, pleated structures. Chymotrypsin contains both alpha-helices and a significant proportion of beta-sheets. These secondary structures form through hydrogen bonds between the backbone atoms of the amino acids.
The Three-Dimensional Shape
The secondary structures of chymotrypsin further fold into a specific three-dimensional arrangement, known as its tertiary structure. This overall shape is often described as a two-domain beta-barrel fold, where two beta-sheets, each composed of six beta-strands, are surrounded by surface helices and loops. This precise folding creates distinct regions within the enzyme.
Several forces stabilize this 3D architecture. Disulfide bonds, formed between the sulfur atoms of cysteine residues, act as covalent links, holding different parts of the polypeptide chain together. Chymotrypsin has both inter-chain and intra-chain disulfide bonds, which maintain its stability. Hydrophobic interactions, where nonpolar amino acid side chains cluster together away from water, also contribute to the enzyme’s stable folded state. Hydrogen bonds and electrostatic interactions further contribute to the structure’s stability.
The Catalytic Core
The active site of chymotrypsin, often called its catalytic core, is a depression on the enzyme’s surface where the chemical reaction takes place. This site contains an arrangement of three amino acid residues: Serine-195 (Ser-195), Histidine-57 (His-57), and Aspartate-102 (Asp-102), collectively known as the catalytic triad. These residues are positioned to work together to facilitate peptide bond cleavage.
Aspartate-102 orients Histidine-57, making it a more effective proton acceptor. Histidine-57 then abstracts a proton from Serine-195, transforming Serine-195 into a reactive alkoxide ion. This alkoxide ion, a nucleophile, attacks the carbonyl carbon of the peptide bond in the substrate, forming a temporary covalent bond and cleaving the peptide bond. The enzyme also contains an “oxyanion hole,” formed by the backbone nitrogens of Serine-195 and Glycine-193, which stabilizes the transient negatively charged oxygen during the reaction.
Chymotrypsin’s specificity in cleaving peptide bonds after large hydrophobic amino acids like tyrosine, tryptophan, and phenylalanine is determined by its S1 specificity pocket. This pocket is a hydrophobic cavity within the active site that accommodates the aromatic rings of these amino acids. The complementary shape and hydrophobic nature of the S1 pocket ensure that only substrates with suitable side chains bind effectively, initiating the catalytic process.
From Inactive to Active Form
Chymotrypsin is not produced in its active form; instead, it is synthesized in the pancreas as an inactive precursor called chymotrypsinogen. This inactive form, or zymogen, prevents the enzyme from digesting the pancreas itself.
Activation occurs when chymotrypsinogen is secreted into the small intestine. The enzyme trypsin initiates this process by cleaving a peptide bond between Arginine-15 and Isoleucine-16 in the chymotrypsinogen molecule. This initial cleavage results in an intermediate active form called π-chymotrypsin.
The cleavage at Arg15-Ile16 triggers a series of conformational changes within the molecule. The newly exposed amino-terminal group of Isoleucine-16 moves inward and forms an ionic bond with Aspartate-194. This interaction, along with other structural rearrangements, leads to the formation of the active site, including the establishment of the oxyanion hole and the S1 pocket. Subsequent self-cleavages by π-chymotrypsin remove two dipeptides, resulting in α-chymotrypsin, which consists of three polypeptide chains held together by disulfide bonds.