Pepsin
Created by Megan Gutierrez
Porcine pepsin is a proteolytic enzyme which resides in the stomach of Sus scrofa. Pepsin's role in the stomach is to digest proteins through the hydrolysis of peptide bonds(1). At the optimal pH of 2, pepsin physiologically exists in monomeric form. At higher pH values of 4.5-6, however, aggregates and possible oligomers have been observed(2). These aggregates have yet to be carefully studied and their structures and properties are unknown. Pepsin is thought to exist in different conformations at pH values conducive to aggregate formation, but the structures of these possible alternate conformations are not well characterized(2).
One subunit of 326 amino acids comprises pepsin, which has a molecular weight of 34,509.83g and an isoelectric point of 3.24(3). Pepsin is classified as bilobal, consisting of two domains which become close in proximity upon proper protein folding. The interaction between Asp-32 and Asp-215, each residue located on a different lobe, leads to formation of the active site(4). Pepsin catalyzes the hydrolysis of peptide bonds between hydrophobic or aromatic residues of a protein substrate. Bonds such as Phe-Phe, Phe-Trp, and Phe-Tyr are commonly hydrolyzed(1). The bilobal structure of pepsin is critical to the formation of the active site and thus to the protein's overall function. The hydrolysis reaction carried out by pepsin contributes greatly to digestive capability and nutrient absorption, enabling organisms to obtain molecules essential for survival.
Pepsin contains elements of secondary structure including alpha helices, beta sheets, and random coils. According to sequence data provided by the Protein Data Bank, pepsin is composed of fourteen percent helices and forty-four percent beta sheets. There are ten alpha helices encompassing forty-six residues and thirty-two beta strands encompassing one hundred forty-four residues(5). Random coils are indicated as well. Secondary structure is crucial in achieving enzymatic function. Two antiparallel beta strands on the surface of the protein containing residues Leu-71 through Gly-82 form a loose, flexible flap. The flap is located near the cleft of the active site and its high degree of motility is hypothesized to allow substrates into and out of the active site, simultaneously shielding the active site from the environment while the reaction proceeds(6). The flexibility and stability conferred through secondary structure lend form and function to pepsin.
Pepsin has several functionally important residues. Of paramount importance are Asp-32 and Asp-215, the residues of the catalytic site. These two residues classify pepsin as an aspartic protease. Asp-32 and Asp-215 are each part of two conserved sequences of Asp-Thr-Gly. These sequences converge to form the catalytic site, bringing Asp-32 and Asp-215 together. The polar nature of aspartate allows these residues to hydrogen bond to water and the substrate in order to correctly position the reagents(7). The acidity of aspartate then functions to cleave the targeted peptide bond of the substrate through a mechanism of general acid-base catalysis. Water plays a crucial role in this mechanism. Asp-32 is temporarily attached to the N-terminal end of the cleaved protein substrate through hydrogen bonds, yet is liberated soon after completion of the reaction(7).
Other residues which contribute to the alignment of the substrate in the active site are Gly-76 and Thr-77, both located in the aforementioned flap region of pepsin. Gly-76 is part of a network of hydrogen bonds which anchors the substrate during catalysis. The inherent flexibility of glycine itself lends an additional function to this residue, the contribution of increased motility to the flap. Mutagenesis studies have demonstrated that this motility plays a role in the speed of the catalysis reaction(8). Like Gly-76, Thr-77 also anchors the substrate near the active site through hydrogen bond formation. The polar nature of threonine, due to the free hydroxyl group, contributes to these hydrogen bonds(6).
Thr-77, however, has another role in the maturation of pepsin. Pepsin is first secreted from the gastric glands of the stomach as pepsinogen (PDB ID=3PSG). Pepsinogen is the zymogen form of pepsin. Pepsinogen differs from pepsin in that it contains an additional forty-four amino acids on the N-terminus of the chain. These extra amino acids are termed the prosegment(6). In pepsinogen, the prosegment interacts with the other amino acids through electrostatic interactions between acidic and basic residues. At the acidic pH of gastric juice, the acidic residues become protonated and lose their charge, decreasing their affinity for basic amino acids, and pepsinogen is destabilized. An undefined autocatalytic process which involves Thr-77 can then occur. Thr-77 is hypothesized to have a role in the proteolytic cleavage which liberates the prosegment and allows pepsin to achieve its native conformation(9).
A conserved lysine residue has an important function in pepsin. Lys-320 is near the C-terminus of the peptide and is located near both the active site and the flap region of folded pepsin. This positively charged residue aids interacts with the flap to influence flap stability and motility, impacting the speed of catalysis(10).
Finally, the residues His-53 and Gln-55 contribute to ligand interaction. Dimethyl sulphoxide is a polar ligand and forms hydrogen bonds with these residues to destabilize and denature pepsin. Water molecules play a role in this destabilization as well(11). The new interactions change pepsin's secondary structure and lead to loss of function. The purpose of introducing DMSO into a pepsin solution is to study the processes which occur during protein denaturation(11).
DMSO is not the only molecule which can interfere with pepsin's activity. Pepsin is known to be inhibited by pepstatin, which interferes with aspartic proteases. Pepstatin is a small molecule derived from the bacterial species Actinomyces and its mode of inhibition has yet to be discovered. Pepstatin was once used in experimental trials to treat gastric ulcers, but was ultimately unsuccessful(12).
Human uropepsin (PDB ID=1FLH) shares many structural features with pepsin, though their functions vary slightly. A BLAST search was conducted to find a protein with similar primary structure to pepsin. An E-score of 7e-163(13) illustrates that uropepsin and pepsin are similar in primary sequence. The proteins share 86.5% sequence similarity. In addition, both have residues Asp-32 and Asp-215 at the catalytic site and are thus aspartic proteases(14). A Dali search was then conducted to find a protein with similar tertiary structure to pepsin. Results indicated that uropepsin was a match once again based on a Z-score of 52.6 and rmsd=0.6A(15). Pepsin and uropepsin share a bilobal structure of mostly beta sheets(14). These similarities in primary and tertiary structures translate to similarity in overall function. Both proteins function to cleave peptide bonds(16), though uropepsin operates in the bloodstream and has exocrine function while pepsin operates only in the stomach(14). It can be concluded that pepsin and uropepsin have similarities in structure and function.