Elastase
Created by Nathan Glass
Pancreatic elastase, isolated from a pig, or Sus scrofa ( PDB ID = 3EST) is an endopeptidase with a molecular weight of 25907.11amu, an isoelectric point at pH=8.65 and a primary structure that is 240 residues long. It belongs to a larger, more diverse family of proteins referred to as serine proteases. This family of proteins is involved in the catalysis of the hydrolysis of peptide bonds, and all of the proteins belonging to this family bear at least one serine residue at their active site (1).
Biologically, serine proteases are important because they break down proteins that could neither be expelled from the body, nor absorbed or used by the body due to their large size. Porcine pancreatic elastase (PPE) in particular is synthesized in the pancreas of a pig and secreted in the intestines; its primary function is to break down elastin, an elastic fiber that works with collagen to give connective tissue its characteristic features (1). When the native PPE protein cannot perform properly, health problems may be met, these problems may include pancreatitis, digestive problems or larger problems involving the build-up of elastin in the connective tissues of an animal due to the unnatural inhibition and/or excitation of elastase.
The secondary and tertiary structure of PPE consists of two antiparallel, beta-barrel, cylindrical domains, referred to as the 'left' and 'right' domains. These domains are rotated by sixty degrees about an axis perpendicular to the barrel vectors (3). This forms what is referred to as the "primary rib cage" of the native porcine elastase. The extended binding site and catalytic active site are found in the crevice between the two domains, and while no disulfide bridge is present to connect these two domains, it has been shown that the crossing of the termini of PPE holds the two domains together.
For this to happen, the Val residue at position 16 of the polypeptide chain crosses over to the 'left' domain and forms a salt bridge with the atom O-2 of Asp-194, at the C-terminus. This provides flexibility and stability that allows for the "induced fit" of substrates at the active site.
During the catalysis of reactions, substrate proteins bind to PPE at its primary active site, which consists of three residues in particular that work in symphony to aid in the hydrolysis of peptide bonds. These residues are His-57, Asp-102 and Ser-195, and they are referred to as the "catalytic triad" in the family of serine proteases. Each residue in the catalytic triad has a specific function. The -OH group of the serine at the active site acts as a nucleophile that attacks the carbonyl carbon of the peptide bond that is to be hydrolyzed. The histidine residue at the active site contains a pair of electrons that aid in the nucleophilic attack of the carbonyl carbon by accepting serine's hydrogen. Additionally, histidine's pair of electrons is made much more electronegative through hydrogen bonding with the carboxyl group of aspartate. These forces lower the activation energy of the hydrolization reaction that is necessary to break substrates' peptide bonds (1) -- the mechanism by through which this works can be seen here. This serves an important role in living organisms in regulating elastin concentrations, and the regulation of elastase itself through the binding of inhibitors, like 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin in the covalent enzyme-inhibitor complex in the example here (4), can have direct physiological effects on a living organism through the overexpression of elastin due to the inhibition of elastase.
In addition to the aforementioned features, PPE also contains three ligands. The first of which is a calcium ion -- while it is known that the calcium ion binds at the " calcium binding site" near the active site of elastase, it is unclear hwat it's exact, biochemical purpose is. It might contribute to the characteristic binding observed in PPE-substrate complexes, but it remains unclear (5). Two sulfate ions constitute the other PPE's two other ligands. One ion is on the exterior of the protein; it is hydrogen bonded to the guanidinium groups Arg-230, while the other sulfate ion is in the active site region, this makes hydrogen bonds with Gly-193 (1).
In comparison to PPE, Salmon pancreatic elastase (SPE, PDB ID = 1ELT) (Superimposed with PPE here) serves the same biological purpose in salmon as PPE does in pigs, but it is somewhat different structurally (2). There is a deletion of the amino acid residue at position 186 of SPE. The residue at position 192, normally glutamine in PPE, is substituted with asparagine in SPE. One of the calcium ligands in SPE is one C-C longer than the one found in PPE; and lastly, the electron density seen in the final three residues of the C-terminus of native PPE is not present in SPE. Biologically, SPE has been reported to hydrolyze certain elastase substrates more efficiently than mammalian pancreatic elastase; SPE has also been shown to be less stable at low pH and more stable at low temperatures than PPE (7). SPE and PPE have remarkably similar primary structures though, and an analysis of tertiary structure was conducted for PPE using the "Dali Server" and SPE was found to have a Z-score of 44.4 and a root-mean-squared deviation of 0.5 angstroms (6). This means that, if the backbones of both proteins were lined up as tightly as possible, there would only be an average distance of 0.5 angstroms between the skeleton-structures. These structural similarities might be accountable for the basic similarites in function that may be observed between PPE and SPE in vivo, but it is still unclear which specific structural differences may be accountable for the experimentally observable differences in function between SPE and PPE (7).