From Proteopedia

Trypsin

(Specifically PDB: 1QLQ)

Overview

Trypsin was first isolated by Wilhelm Kühne in 1867^[1]. Trypsin is a serine protease synthesized in the pancreas but is not activated until the zymogen form of trypsin is activated. This prevents trypsin from digesting actual body tissue^[2]. Serine proteases were instrumental in the discovery and subsequent study of enzymes due to there high stability and large quantities in digestive juices. One of the first proteins to be studied via X-ray crystallography was Chymotrypsin. Trypsin cleaves on the C-terminus side of lysine and arginine^[3]. An easy way to distinguish between main structural components of the protein is to view it using

Structure

Trypsin's primary amino acid sequence (RPDFCLEPPYAGACRARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCLRTCGGA) ^[4] forms the of the protein, which then folds into secondary structures, consisting of two and two . Both of the α helices are right handed and the β sheets are anti-parallel. The order of the secondary structures is easily visible when using the scheme to identify secondary structures. The N-terminus (blue) is the beginning of trypsin and the C-terminus (agua-green) is the end.

Polar and Nonpolar Residues

Polar residues are typically hydrophobic, and seek to be sheltered from the aqueous environments that proteins typically inhibit. The polarity of an amino acid is determined by its (orange). When considering the it may look like the polar (blue) and nonpolar (crimson) residues are not organized in a specific manner, but when you consider the it is evident that the majority of the nonpolar residues are shielded by the polar residues. Another way to show this principle is by looking at the location of the of Trypsin (red). Alternatively, for a more in depth analysis of trypsin, you can view which can be even more informing. The hydrophobic portions desire to be shielded from the water in the smallest area possible in order to minimize its interaction with water, thereby maximizing the entropy of the water. It is evident that basically all water molecules are kept outside the protein when viewing a (water-blue, trypsin-orange). This form of trypsin (PDB 1QLQ), has been modified to help enable its crystalization, and thus has four water molecules inside of it instead of the normal three which is present in the wild-type trpsin^[5].

Intramolecular and Intermolecular Forces

The structure of trypsin is stabilized by a variety of intramolecular and intermolecular forces. Trypsin has three which form between the cysteine amino acids. The cysteine amino acids are shown as pink, and you can see how they are placed in the proper 3-dimensional space to bond with each other. The bond they form is represented by the yellow bar in between them, as each of their sulfurs bind to one another. Disulfide bonds are especially important for structural stability in extracellular environments, where conditions are more prone to fluctuation. Secondary structures are stabilized via interactions that compliment their specific side chains. For example, the first α helix in trypsin's structure is stabilized by several other in the molecule itself. The ball and stick amino acids marked with an * are part of α helix while the space filling molecules stabilize the α helix. the The α helix is also stabilized by as well as (water is dark blue). The (β sheets are ball and stick) have a more bilaterally divided type of bonding. One side of the β sheets are exposed to water (pink), and are stabilized by hydrogen bonding. Additionally, there are many hydrophobic interactions (gray) on the internal side of the β sheets. There are some intramolecular hydrogen bonding which is shown as light blue(oxygen) and blue(nitrogen).

Ligands

There are four present in 1QLQ, which are stabilized mostly by hydrogen bonding. For example, is stabilized by hydrogen bonds using the oxygens on SO4. There is a image to the left showing the bonding interaction.

Cleavage Mechanism

Serine proteases cleave using what is commonly called a catalytic triad. This catalytic triad consists of Asp 102, His 57, and Ser 195^[6]. The cleavage mechanism is shown to the left. First, the substrate binds to trypsin, and then the side chain oxygen of Ser 195 nucleophilicly attacks, with assist from His 57. Next, the peptide bond is cleaved, with His 57 assisting again with stabilization. After cleavage, the first product is released. Next there is a nucleophilic attack of H20 on the acyl-enzye intermediate (assistance of His 57). This is followed by the decomposition of the acyl intermediate and release of the second product^[7]. You are able to view the actual binding site.

Trypsinogen

Trypsin's zymogen form is called trypsinogen, and can actually activate itself. Zymogens require a biochemical change to activate. Only an activated form of trypsin can activate the trypsinogen, and this initial activation is carried out by enteropeptidase, which is a serine protease as well. Because activated forms of trypsin can activate others, trypsin is said to be autocatalytic^[8]. In addition to activating itself, it can also activate chymotrypsin and elastase.

References

1. ↑ ISBN 952-10-1863-1
2. ↑ doi:10.1016/j.theochem.2003.08.072
3. ↑ Protein Data Bank
4. ↑ 1qlq
5. ↑ Czapinska, Honorata et al. "High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence." Journal of Molecular Biology. Volume 295, Issue 5, 4 February 2000, Pages 1237-1249 doi:10.1006/jmbi.1999.3445
6. ↑ Polgár L. "The catalytic triad of serine peptidases". Cell. Mol. Life Sci. October 2005. 62 (19-20): 2161–72. doi:10.1007/s00018-005-5160-x
7. ↑ Polgár L. "The catalytic triad of serine peptidases". Cell. Mol. Life Sci. October 2005. 62 (19-20): 2161–72. doi:10.1007/s00018-005-5160-x
8. ↑ Voet, Donald et al. Fundamentals of Biochemistry - Life at the Molecular Level. 3rd ed. John Wiley & Sons, Inc. 2008